Keywords

1 Learning Objectives

  • An overview of the contrasting genetic and molecular interactions that occur between wheat and its pathogens.

2 Pioneering Studies in Model Biotrophic Pathosystems

Plant diseases reduce crop yield potential. Resistant cultivars provide the most cost effective and environmentally friendly means for disease control. A major ongoing problem with disease resistance has been that once widely deployed, its effectiveness is lost due to genetic changes in the respective pathogens. Consequently, an ongoing search for ever-more host resistance genes is required. The problem of resistance gene failure has become so significant that in some countries, growers routinely use fungicides and pesticides, which can lose effectiveness and also cause potential health and environmental issues.

The challenge in using genetically conferred disease resistance is therefore to find ways of prolonging the useful period (‘durability’) of a particular resistance source. This requires a thorough understanding of the genetic interactions between the host plant and pest/pathogen system. A major advance was made in the biotrophic flax plant/flax rust pathosystem by Flor in the 1950s. Biotrophic pathogens like rust diseases can only grow on living plant tissue in contrast to nectotrophic pathogens, which live on dead or dying host tissue (described below). Flor developed the gene-for-gene hypothesis, which states that for every plant resistance gene there is a corresponding gene, encoding an avirulence gene product, in the pathogen that is recognised [1]. Modern molecular biology has confirmed Flor’s insightful research and shown that when pathogens infect their hosts they secrete an array of effector molecules into the plant. Plant hosts have evolved specific receptor molecules, encoded by resistance genes, that each directly or indirectly recognise a particular pathogen effector molecule (i.e. the molecular basis for the gene-for-gene hypothesis). Upon recognition of a pathogen molecule the resistance protein activates a resistance response called effector triggered immunity (ETI). Loss or change in recognised pathogen effectors, which are also called avirulence proteins, leads to no pathogen recognition by the plant and hence a loss of resistance to biotrophic pathogens. Changing or losing recognised avirulence effector molecules is how new races of pathogens evolve to overcome plant resistance.

Plants also possess a second type of resistance called PAMP (pathogen associated molecular pattern)-triggered immunity (PTI). PTI differs from ETI in that all microbes possess some conserved molecules (e.g. chitin in fungal cell walls, bacterial flagellin protein) that the plant can recognise with pattern recognition receptors (PRRs) leading to the activation of PTI. This resistance protects plants against most potential biotrophic pathogens. For biotrophic pathogens, such as wheat rusts and mildews, to be able to grow on a particular plant species it must suppress the PTI response of the host, which it does by introducing effector molecules. The suite of effectors that each pathogen possesses and the ability of these molecules to effectively target specific plant proteins plays a large role in determining what plant species the pathogen can colonise. As described above, plants in turn have evolved R proteins that can recognise specific effectors and activate ETI, thereby making the host resistant. In turn the pathogen loses or alters recognised effectors in an ongoing arms race between the host and the pathogen [2].

Flor worked on flax rust, a disease caused by an autoecious, dikaryotic fungus that infects flax, a self-pollinating diploid host plant. A detailed knowledge of the life cycle and breeding behaviour of each organism and an ability to perform genetic crosses on both were essential for his discoveries. Flor’s discoveries were not only applicable to the flax/flax rust system but also held true in many other pathosystems as well. He established two basic principles, firstly, the genetic interaction that led to incompatibility (i.e. resistance in the host and avirulence in the pathogen) involved dominant genes in both the plant and the pathogen. Dominance is a strong indicator of a functional gene in contrast to recessive, often loss of function mutations. Secondly, genetic knowledge of one organism enabled the genotype of the other to be determined. This is the genetic basis of pathogen race surveys where isolates are screened against a panel of plants with known resistance genes, enabling what avirulence and virulence genes are present in the pathogen to be determined. This information in turn informs about the resistance or susceptibility of elite wheat cultivars, that contain known resistance genes, to each pathogen isolate. It also provides information on the genetic relationships and evolutionary pathways existing between different pathogen races.

Advances in molecular genetics over the last 25 years (50 years post-Flor) have enabled gene cloning in plant and pathogen species, which has confirmed Flor’s work. In the case of rust disease resistance, a maize transposable element (Ac) was used to generate insertional mutants of flax rust resistance genes thereby enabling their cloning (see Fig. 19.1). The molecular structure of several rust resistance genes and their products was then determined. Transposon tagging was also used to identify the tobacco mosaic virus N resistance gene in tobacco and maize Rp1 rust resistance gene, while map-based cloning, which uses linked DNA markers as entry points to scan overlapping large DNA fragments to identify gene candidates (Fig. 19.1), enabled the isolation of the Arabidopsis RPS2 bacterial resistance. From these studies which used fungal, viral and bacterial species, respectively, the flax rust L6, tobacco N and Arabidopsis RPS2 resistance genes were shown to encode proteins with a similar modular structure of an N-terminal nucleotide binding site and C-terminal leucine rich repeats (NLR) [3].

Fig. 19.1
figure 1

Classic methods of resistance gene isolation. Transposon tagging using heterologous transposons was used to isolate a number of resistance genes such as the flax L6 gene, tobacco N gene and maize Rp1 gene. Susceptible mutants arising from transposon insertions in the causative R gene were sought from active transposon lines. The transposon insertion then acted as a molecular tag to enable isolation of the R gene. An alternative approach was map-based cloning where markers closely linked to an R gene were sought. These markers then enabled the isolation of large DNA fragments from the locus by screening large insert BAC, PAC and YAC libraries. Overlapping DNA inserts that spanned the locus were then sought and analysed for R gene candidates. Technology advances have created new methods of R gene isolation based on exome capture or chromosome isolation as detailed in Table 19.1

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NLR genes were subsequently identified in all plant species and shown to be the largest class of disease resistance genes present in plants, including wheat. The current pan genome of bread wheat, which is derived from 16 cultivars, contains around 2500 NLR genes, 31–34% of which are shared across all genomes. The number of unique NLR’s ranges from 22 to 192 per cultivar [4]. The NLR gene family is highly diverse, although some genes appear orthologous across species as well as homoeologous within some of the Triticeae species. NLR proteins function in two ways, by either directly recognising a single specific pathogen effector molecule introduced into the plant cell upon which ETI is activated, or alternatively, by recognising the modification of a host protein targeted by a pathogen effector (guard model) again leading to ETI [5]. After effector recognition by an NLR protein a complex defense cascade is activated that can be accompanied by host cell hypersensitive cell death in some instances.

3 Genetics of Resistance to Wheat Biotrophic Pathogens- Rusts and Mildew

Increasing sophistication and advances in both genomic and marker technologies (Table 19.1) have led to a rapid increase in the cloning of powdery mildew and rust (see Chap. 7) resistance genes from the complex wheat genome (Table 19.2). The majority of these genes are race-specific all plant developmental stage resistance (ASR) genes that usually encode NLR proteins (Table 19.2). In general, each resistance specificity is conferred by a single NLR gene at the locus although the genomic organisation of NLR loci is variable. The wheat Pm3 locus consists of a single gene that encodes at least 56 allelic sequences of which 17 have been shown to be functional [6] (examples shown in Table 19.2). The proteins encoded by these alleles share at least 97% identity. This gene is therefore analogous to the barley Mla powdery mildew locus, the Arabidopsis RPP13 downy mildew resistance locus and flax L resistance locus which all encode large allelic series. However, most cloned wheat rust and mildew resistance genes are members of small, tandem gene families with a single member conferring the resistance phenotype. In some instances, orthologous sequences in different species have been shown to encode functional resistance. For example, orthologues of the barley Mla gene present in T. monococcum, Secale secalis and Ae. tauschii encode the TaMla1, Sr50 and Sr33 resistance genes, respectively [7].

Table 19.1 Marker and cloning technologies used in wheat for mapping and isolation of resistance genes
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Table 19.2 Examples of cloned wheat resistance genes and their corresponding pathogen effectors molecules (modified with permission from [8])
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Both single gene, multi-allele loci and tandem gene families evolve new resistance specificities by intra or intergenic recombination to encode new protein sequences. Central to this recombination process is sequence diversification arising from mutation and selection favouring protein diversification, rather than conservation, at specific regions in the NLR protein. These mechanisms enable new plant resistance specifies to evolve which are required to combat pathogen populations that can rapidly evolve new virulences by loss or alteration of avirulence genes.

An exception in wheat to the generalisation that only a single NLR protein is required for resistance activation is the Lr10 locus where two sequence unrelated NLR genes are needed to give resistance [9]. Resistance loci that encode dual NLR genes required for resistance have been identified in other plant species [5]. In some of these dual NLR systems one protein contains an “integrated decoy” domain that interacts directly with a pathogen effector. The decoy domain shows similarity to other plant proteins that are likely the true host targets of the effector [5]. The second NLR member contributes to signalling a defense response upon effector recognition by the first member [5]. Approximately 10% of plant NLR genes encode integrated decoy domains [10] and 28 different integrated domains have been identified in wheat NLR genes. However, an integrated decoy domain has not been identified in either NLR gene required for Lr10 resistance.

As described above, during biotrophic pathogen colonisation large numbers of effector proteins are introduced into host cells to suppress plant defence responses and alter cell homeostasis to benefit the pathogen [11]. Each effector targets particular plant proteins or processes within the plant cell. The genomes of the wheat powdery mildew pathogen, Blumeria graminis f. sp. tritici (Bgt) and wheat rust pathogens Puccinia triticina (Pt) and P. graminis f. sp. tritici (Pgt) encode in excess of 800, 2000 and 1700 predicted, secreted effector protein genes, respectively. The function of most effectors and their host targets is unknown, however, some wheat pathogen effectors have been shown to target transcription factors, mRNA processing apparatus, chloroplasts and suppress plant defense responses. Similar to plant resistance genes, diversifying selection also occurs in effector genes.

For both wheat stem rust and powdery mildew several pathogen avirulence genes and their cognate resistance genes have been cloned (Table 19.2). Of the two wheat stem rust effector genes cloned, AvrSr35 and AvrSr50, both encode proteins that directly bind to their corresponding resistance proteins, Sr35 and Sr50, respectively [12, 13]. Similarly, some mildew effector proteins have also been shown to directly bind to their corresponding NLR protein encoded by the barley Mla locus. However, in many other pathosystems this is not the case and these effectors are assumed to target and modify host proteins, with the R protein then recognising the modified host protein that it guards. While Sr35 and Sr50 recognise single effectors (AvrSr35 and AvrSr50, respectively) some members of the Pm3 allelic series can recognise multiple effectors (Table 19.2).

However, not all wheat ASR genes encode NLR proteins with Yr15 and Sr60 being exceptions. Both genes encode tandem protein kinases [14, 15] making them structurally distinct and their mode of action has yet to be determined. The Yr15 and Sr60 proteins are structurally similar to the barley stem rust resistance protein, Rpg1, which is a dual kinase protein, albeit with one kinase domain no longer functional.

Several APR genes have also been cloned from wheat. Both the Lr34/Yr18/Sr57/Pm38/Ltn1 APR gene (hereafter Lr34) and the Lr67/Yr46/Sr55/Pm46/Ltn3 APR gene (hereafter Lr67) provide broad spectrum resistance to Pt, Pst, Pgt and Bgt pathogens [16, 17]. Interestingly both genes also induce premature senescence of mature leaf tips (leaf tip necrosis (ltn)) suggesting a mechanistic commonality. Consist with this hypothesis is the lack of additivity of these two partial adult plant resistance genes. However, the products of these two genes are different. Lr34 encodes an ABC transporter protein that is suggested to transport abscisic acid while Lr67 encodes a hexose transporter protein no longer capable of sugar transport.

How these two genes provide resistance has not yet been fully established; however, a remarkable feature of Lr34 is its durability having been used in agriculture for many decades without being overcome. A third wheat gene, Yr36, is unlike Lr34 and Lr67 in that it confers partial APR to Pst only. Yr36 encodes a START domain containing kinase protein that is believed to interact with a chloroplast peroxidase protein resulting in elevated levels of hydrogen peroxide production [18]. Interestingly Yr36 shows additive resistance with both Lr34 and Lr67 suggesting a different mode of action to these other two genes.

However, not all APR genes are broad-spectrum. A number of race-specific genes (e.g. Lr12, Lr22a, Lr22b, Yr49) have been identified that do not provide resistance until later in plant development [19]. One of these genes, Lr22a, has been cloned and shown to encode an NLR protein [20]. Given the tendency of NLR genes to be overcome by pathogen mutation to virulence it seems unlikely that this latter type of APR will remain durable.

4 Genetics of Resistance to Wheat Necrotrophic Pathogens

Necrotrophic pathogens (see Chap. 8) differ from biotrophic pathogens, like rusts (see Chap. 7) and mildews, in that they require dead or dying cells to acquire nutrients, whereas biotrophs require living plant cells to proliferate. Initially it was thought that necrotrophs exuded a barrage of cell wall-degrading enzymes to kill their host and allow them to acquire nutrients. However, research over the past two decades on interactions between wheat and necrotrophic fungal pathogens Pyrenophora tritici-repentis and Parastagonospora nodorum has shown that more complex mechanisms are involved. In these pathosystems, pathogen-produced effectors, which are called necrotrophic effectors (NEs) (formerly called host-selective toxins) are recognized by the products of specific plant genes in an inverse gene-for-gene manner (Fig. 19.2). In other words, recognition of a fungal NE by the product of a dominant host gene leads to a compatible interaction (disease susceptibility), and the lack of recognition of the pathogen leads to resistance. Therefore, in plant-necrotroph interactions, plant genes that actively recognize the pathogen are considered susceptibility genes as opposed to plant-biotroph interactions where they act as resistance genes (Fig. 19.2) [21].

Fig. 19.2
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Gene-for-gene interactions occurring between plant hosts and biotrophic pathogens and inverse gene-for-gene interactions with necrotrophic pathogens. (Top left panel) When a biotrophic pathogen introduces a recognised effector (avirulence effector) into a plant cell containing the corresponding resistance gene a defense response is activated that inhibits further pathogen development and can lead to programmed cell death of the infected cell. (Bottom left panel) Conversely if the host plant lacks the appropriate resistance gene or the biotrophic pathogen lacks the recognised effector plant disease susceptibility occurs. (Top right panel) In contrast, in necrotrophic interactions a recognised pathogen effector leads to plant cell death that is required for pathogen development. In the absence of the appropriate pathogen effector or plant resistance protein, which in this case is a susceptibility factor, resistance to the necrotrophic pathogen occurs

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The first NE to be identified from a wheat pathogen was Ptr ToxA, which was first discovered in the foliar disease tan spot pathogen Pyrenophora tritici-repentis and later in pathogens that cause Septoria nodorum blotch (SNB) (Parastagonospora nodorum) and spot blotch (Bipolaris sorokianiana). ToxA is a 13.2 kDa protein encoded by a single gene and was the first proteinaceous NE identified in a plant pathogen [22].

Early work in the wheat-tan spot system demonstrated that ToxA is a significant virulence factor and that host resistance is governed by a single recessive gene on chromosome 5BL designated Tsn1 [23]. Subsequent work evaluating Chinese Spring nullisomic-tetrasomic stocks and chromosome deletion lines demonstrated that resistance was not governed by an actively expressed recessive gene, but rather the lack of a dominant susceptibility gene, as plants null for chromosome 5B were insensitive to ToxA. This finding showed that pathogen recognition by the host plant was necessary to confer susceptibility, and all wheat gene-NE interactions in the wheat-Pa. nodorum and wheat-Py. tritici-repentis pathosystems have since been found to operate in the same manner.

Map-based cloning of Tsn1 [23] showed it encodes an NLR similar to typical plant disease resistance proteins that recognize biotrophic pathogens, but it also contained a serine/threonine protein kinase (PK) domain (Table 19.2). Only the barley stem rust resistance gene Rpg5 has been shown to encode an NLR with an integrated PK domain. The two genes differ in that the PK domain is located at the N terminus in Tsn1 and at the C terminus in Rpg5 indicating that they arose through independent gene fusion events.

Tsn1 does not physically interact with ToxA directly but rather interacts with a chloroplast-localized plastocyanin protein called ToxABP1 and a pathogenesis-related protein 1 (PR-1) [22], suggesting that Tsn1 is a guard of ToxABP1, as described above in the guard hypothesis. The Tsn1-ToxA interactions requires light to manifest necrosis because Tsn1 transcription is strongly downregulated under darkness. There is also evidence that a compatible Tsn1-ToxA interaction leads to photosystem alterations. These findings suggest that ToxA recognition by Tsn1 leads to disruption of photosynthesis. However, a compatible Tsn1-ToxA interaction also leads to host responses typically observed in resistance to biotrophic pathogens including production of phenylpropanoids and reactive oxygen species, lignification and electrolyte leakage, which indicates hijacking of ETI pathways [21].

ToxA is functionally redundant in the three pathogen species that it has been identified in and acquisition of the ToxA gene likely occurred through interspecific gene transfer. However, the level of virulence conferred by ToxA can vary depending on the pathogen and the host genetic background. For the disease SNB caused by Pa. nodorum, the Tsn1-ToxA interaction plays a major role in susceptibility of both durum and common wheat, but for tan spot caused by Py. tritici-repentis, the same interaction has never been associated with disease susceptibility in durum wheat and its contribution to tan spot virulence in common wheat can range from nothing to major depending on the host genetic background. More studies are needed to determine the potential variability of the Tsn1-ToxA interaction in the wheat-B. sorokianiana pathosystem.

In addition to the Tsn1-ToxA inverse gene-for-gene relationship in the wheat-Pa. nodorum pathosystem, eight additional sensitivity genes that recognize proteinaceous pathogen-produced NEs to confer SNB have been identified [22]. These include the wheat genes Snn1, Snn2, homoeologous genes Snn3-B1 and Snn3-D1, Snn4, Snn5, Snn6, and Snn7. Snn1, Snn4, and Snn5 reside on chromosome arms 1BS, 1AS, and 4BL, and recognize the NEs SnTox1, SnTox4, and SnTox5, respectively. Snn3-B1 and Snn3-D1 are homoeologous genes located on 5BS and 5DS, respectively, and both recognize the NE SnTox3. Genes Snn2, Snn6, and Snn7 are located on 2DS, 6AL, and 2DL, respectively, and were originally thought to recognize different NEs (SnTox2, SnTox6, SnTox7). However, it was recently shown that these three NEs were actually the same molecule and therefore designated as SnTox267. Among these wheat genes, Snn1 and Snn3-D1 have been cloned as have their corresponding NE-producing genes SnTox1 and SnTox3.

The Snn1 gene was the first wheat gene identified to confer sensitivity to a Pa. nodorum NE, and the Snn1-SnTox1 interaction plays a significant role in the development of SNB in wheat. SnTox1 is a 10.3 kDa protein that facilitates infection of mesophyll cells, and also protects the fungal mycelium from plant-produced chitinases by binding chitin [22]. Therefore, SnTox1 serves a dual function, which provides an explanation as to why the SnTox1 gene is highly prevalent among isolates.

Snn1 was isolated from Chinese Spring by positional cloning [24] and encodes a wall-associated kinase (WAK), which is a receptor kinase class that harbor intracellular PK domains and extracellular galacturonan binding (GUB_WAK) and epidermal growth factor-calcium binding (EGF_CA) domains. WAKs usually serve as PRRs that recognize pathogen or damage-associated molecular patterns (PAMPs or DAMPs) leading to PTI. Whereas the WAK gene Snn1 confers SNB susceptibility, recent research has revealed that other biotroph or hemibiotroph resistance genes are also members of the WAK class of receptor kinases. Therefore, whereas ToxA hijacks a Tsn1-associated ETI pathway, SnTox1 hijacks a PTI pathway thus revealing the capability of necrotrophic fungal pathogens to subvert diverse host resistance pathways for pathogenesis.

The SnTox1 protein interacts directly with the extracellular portion of the Snn1 protein [24]. Upon recognition, the mitogen-activated protein kinase (MAPK) gene TaMAPK3 is rapidly upregulated, which is followed by an oxidative burst, DNA laddering, PR gene expression, and cell death, all of which are hallmarks of a defense response to biotrophic pathogens. Like Tsn1, Snn1 transcription is tightly light regulated and a compatible Snn1-SnTox1 interaction is light-dependent suggesting again that photosynthetic pathways are likely involved.

The Snn3 gene recognizes the Pa. nodorum NE SnTox3, which leads to SNB susceptibility. SnTox3 is a 25.8 kDa protein that does not interact directly with the Snn3 protein but, like ToxA, does interact with PR-1 protein family members. Like SnTox1, SnTox3 appears to also have a dual function by hijacking a host necrosis-inducing pathway through the activation of Snn3 and suppressing PR-1-mediated defenses [25].

The identification of Aegiliops tauschii accessions that were sensitive to SnTox3 led to the mapping of the SnTox3-sensitivity gene on chromosome arm 5DS. Comparative mapping experiments revealed that the 5BS and 5DS SnTox3 sensitivity genes were homoeologous, and henceforth referred to as Snn3-B1 and Snn3-D1, respectively. The Ae. tauschii Snn3-D1 gene was subsequently isolated by positional cloning and found to encode integrated PK and major sperm protein (MSP) domains [26]. Therefore, the cloning of a third Pa. nodorum NE sensitivity gene revealed a third class of gene targeted by the pathogen to induce necrosis. Although genes with MSP domains exist in dicots, genes with integrated PK and MSP domains are specific to monocots [26]. An MSP domain was recently shown to be present in the orange blossom wheat midge resistance gene Sm1 where it occurred along with PK and NLR domains. However, the function of MSP domains in plants is yet to be determined.

Three necrotroph susceptibility genes have now been cloned from wheat, and although they all represent a different class of gene, they all harbor a PK domain. It is likely that the PK is necessary for signaling to induce the development of necrosis. The other domains (i.e. NLR, GUB_WAK, EGF_CA, MSP) are likely necessary for NE recognition either directly as is the case with Snn1-SnTox1, or indirectly as observed with Tsn1-ToxA and Snn3-SnTox3. The cloning of additional NE sensitivity genes will shed light on whether additional gene classes are targeted by necrotrophic pathogens.

The wheat-Pa. nodorum pathosystem includes at least nine host gene-NE interactions mentioned above, and the wheat-Py. tritici-repentis pathosystem has three known interactions, i.e. Tsn1-Ptr ToxA, Tsc1-Ptr ToxC, and Tsc2-Ptr ToxB [22]. In addition to these inverse gene-for-gene interactions, numerous resistance QTL have been identified in both systems, and it is unknown whether some QTL may represent additional NE sensitivity genes. In the wheat-Pa. nodorum pathosystem, no qualitative dominant resistance has been identified. However, a single dominant tan spot resistance gene, designated Tsr7, which gives broad-spectrum, race-nonspecific resistance has been identified [27]. Tsr7 was discovered in wild emmer wheat but confirmed to be present in several modern wheat varieties and provide high levels of tan spot resistance. The mechanisms underlying Tsr7-mediated resistance are unknown, but it’s cloning will provide more insights into the molecular interactions between wheat and necrotrophic pathogens.

Tsr7 is a good target for breeding tan spot resistant wheat varieties, but, in addition breeders should also focus on removing NE sensitivity genes for both P. nodorum and Py. tritici-repentis NEs to obtain resistance. However, removal of NE sensitivity genes may come with a couple of caveats. First, because NE sensitivity genes are ‘resistance gene-like’ they may also confer resistance to a biotrophic pathogen as well as susceptibility to a necrotrophic pathogen. So far, there is no indication that any of the identified wheat NE sensitivity genes also provide resistance to any modern races of biotrophic pathogens, but it is not without precedence. The oat Vb gene, which conditions susceptibility to the necrotrophic pathogen that causes Victoria blight, and the Pc-2 gene, which gives resistance to oat crown rust, have never been separated despite much effort suggesting they may be the same gene [28]. Second, breeders must be careful not to inadvertently introgress necrotroph susceptibility genes when breeding for biotrophic resistance. Germplasm breeding material should be characterized to know what susceptibility genes are present, and which molecular markers (see Chap. 28) are used to eliminate, or select against, NE sensitivity genes.

5 Genetics of Resistance to Hemi-Biotrophic Pathogens

Resistance genes to the foliar disease Septoria tritici blotch (STB) caused by the apoplastic fungus Zymoseptoria tritici (see Chap. 9) have been categorized into two groups. Qualitative, race-specific Z. tritici resistance genes with strong phenotypic effects have been reported for over 20 genes. Similar to rust ASR genes, many qualitative STB resistance genes are independent of plant growth stage. By contrast, the quantitative class of resistance genes generally show small to moderate effect and over 80 QTL have been described. An STB gene originating from Aegilops tauschii, Stb16q, was assigned to the quantitative class, however it is one of the few genes that are effective at the seedling stage with major effect against all Z tritici isolates tested. Its classification as a quantitative gene has been called into question and there are suggestions that it be designated as a qualitative gene.

Of the 22 catalogued Stb genes, two have been cloned by positional cloning, namely Stb6 and Stb16q. Stb6 exhibits a ‘gene-for-gene’ relationship and encodes a WAK protein that detects the presence of a matching apoplastic effector, AvrStb6, from Z. tritici. Stb16q encodes a 684 amino acid cysteine rich receptor kinase (CRRK) protein with two extracellular copies of a domain with unknown function (DUF26) characterised by conserved C-X8-C-X2-C motifs, a predicted transmembrane domain and an intracellular serine/threonine (Ser/Thr) protein kinase domain [29]. The DUF26 domain of the Gingko biloba Gnk2 protein shows mannose binding activity suggesting STB16q may therefore recognize apoplastic plant or fungi-derived mannose or derivatives to activate broad-spectrum defense (Saintenac et al. 2020).

Another hemi-biotrophic fungal pathosystem of significance in wheat is Fusarium graminearum which causes head blight (Fusarium head blight-FHB; bleaching of the spike, also referred to as scab) (see Chap. 9). In addition to FHB’s effects on yield loss its ability to contaminate grains with trichothecene mycotoxins pose significant human health problems. Genetics of FHB resistance has largely been reported as quantitative due to the complicated process of infection, confounding plant morphological attributes, plant phenology and growth conditions. Over 200 FHB QTL have been reported in wheat with Fhb1, the first documented and most studied major quantitative trait. From three independent high resolution genetic studies a cluster of genes were identified at the Fhb1 locus. Among the Fhb1 candidate genes, Rawat et al. [30] concluded a pore-forming toxin-like gene as the causative gene while Su et al. [31] and Li et al. [32] reported a histidine-rich calcium-binding protein as the Fhb1 gene product but disagreed about the mode of action. Notwithstanding these contradictory findings, which require clarification, molecular markers generated from the Fhb1 locus have been excellent tools for selecting Fhb1 resistance.

A much clearer definition of the gene underlying FHB resistance in plants carrying Fhb7 derived from the wheat grass species, Thinopyrum elongatum (E genome), has been described [33]. Fhb7 was introgressed into wheat as 7E/7B and 7E/7D chromosomal translocations and subsequently shown to encode a glutathione S-transferase (GST) encoding gene that also conferred crown rot resistance. Fhb7 detoxifies deoxynivalenol (DON- trichothecene toxin) by enzymatic conjugation of a glutathione (GSH) residue onto the toxic epoxide moiety of DON. Unexpectedly Fhb7 appears to originate from the fungal species Epichloë aotearoae and has been transferred to Th elongatum by horizontal gene transfer, i.e. a natural fungus-to-plant gene acquisition. What remains unknown is why Epichloë aotearoae evolved a DON detoxification gene? It has been suggested that it may detoxify the fungus’s own toxins or help it to compete against Fusarium species for grass colonization.

6 Resistance Gene Stacks-Progress Towards Durable Resistance

As already indicated, obtaining durable disease resistance in wheat to rust and mildew pathogens is difficult because ASR genes are often overcome when deployed singularly due to rapid pathogen effector evolution resulting in virulence. In addition, while some APR genes are both durable and broad-spectrum, individual APR genes don’t provide agronomically acceptable levels of disease protection. An obvious solution is to combine multiple ASR genes and/or multiple additive APR genes. However, maintaining polygenic disease resistance combinations throughout breeding programs is an expensive and laborious task, despite modern molecular marker technologies (see Chap. 28).

Genetic engineering offers a potential solution to this problem by enabling multiple cloned disease resistance genes to be introduced into a single locus in the wheat genome. We have recently developed a transgene cassette that encodes four ASR to wheat stem rust and one broad spectrum APR gene i.e. (Sr22/Sr35/Sr45/Sr50/Lr67) [34]. This large gene cassette (approximately 40 kb in size) has been successfully introduced into wheat using Agrobacterium-mediated transformation and transgenics selected that contain all 5 genes at a single locus. In field trials these wheat lines have shown very high levels of disease resistance and additional experiments have confirmed that at least 4 of the 5 genes are functional.

Transgene cassettes therefore offer possibilities to improve resistance durability in wheat because for virulence evolution many pathogen isolates will require multiple mutations to overcome the multiple ASR genes assembled at the locus. A similar approach has also been undertaken in potato where three ASR genes were combined that each provide isolate specific resistance to the potato late blight pathogen Phytophora infestans [35]. Future gene stacks also offer the possibility of combining additive APR genes at a single locus to develop high levels of polygenic APR that can be bred with single locus inheritance.

Another advantage of gene stacking technology is it can help prevent single gene deployment of ASR genes which can lead to their rapid breakdown. Conversely, however, if single gene deployment of members of a gene stack occurs it has the potential to erode the polygenic resistance at this locus. Ideally ASR genes in a gene stack would not have previously been deployed in wheat and be broadly effective against most pathogen isolates. Finally, an obvious impediment to the use of resistance gene stacks in the immediate future is that they are a GM solution. However, a recent amendment in the US ruling that cisgenic transgenics can be considered nonGM may create future opportunities (US Dept Agriculture, 2020).

7 Key Concepts

An ongoing challenge with disease resistance when widely deployed is the general loss of its effectiveness. Accordingly, an ongoing search for new host resistance genes is required. Both single gene, multi-allele loci and tandem gene families evolve new resistance specificities by a variety of mechanisms outlined in the chapter to produce new resistance proteins. These new host resistance specificities play an important role in combating pathogen populations that can rapidly evolve new virulences via loss or alteration of avirulence genes. In plant-necrotroph interactions, plant genes that actively recognize the pathogen are considered susceptibility genes in contrast to plant-biotroph interactions where they serve as resistance genes; a key concept that underlies ‘gene-for-gene’ and ‘inverse gene-for-gene’ relationships.

8 Conclusion

Advances in wheat genome and pan-genome sequencing, mutational genomics and gene capture are facilitating the discovery of resistance genes that are effective against biotrophic and hemibiotrophic pathogens, but act as susceptibility factors in necotrophic pathogen infections. More efficient gene capture technologies are likely to be developed combined with comprehensive pan genome sequences which will further facilitate mutational genomics strategies such as MutRenSeq in isolating resistance genes encoded by NLR immune receptors. Such technologies are likely to broaden beyond NLR’s to include other resistance gene classes such as receptor kinases. These isolated genes will continue to provide perfect markers for breeders and enable further gene stacks to be developed. While gene stacks offer a potential solution for biotrophs and hemi-biotrophs, their applicability to necrotrophic pathogens is far less given the inverse-gene-for-gene relationship; unless more dominant necrotrophic genes like Tsr7 can be found. Other options are likely to include gene editing (see Chap. 29) for rapid knockout of necrotrophic susceptibility genes in elite cultivars. Ultimately these translational research outputs will provide precision breeding using resistance gene toolkits which will augment durable disease resistance strategies.