Innate nonhost immunity in barley to different heterologous rust fungi is controlled by sets of resistance genes with different and overlapping specificities

We developed an evolutionary relevant model system, barley-Puccinia [corrected] rust fungi, to study the inheritance and specificity of plant factors that determine to what extent innate nonhost immunity can be suppressed. A mapping population was developed from a cross between an experimental barley line (SusPtrit) [corrected] with exceptional susceptibility to several heterologous [corrected] (nonhost) rust fungi and regular, immune, cv. Vada [corrected] Seedlings were inoculated with five heterologous [corrected] and two homologous (host) species of rust fungi. Resistance segregated quantitatively for each of the rust fungi. In total, 18 chromosomal regions were implicated. For each rust species, a different set of genes was effective. Of the 18 chromosomal regions, 11 were significantly effective to only one rust species and 7 were effective to more than one rust species, implying genetic linkage or pleiotropy. One resistance (R) gene for hypersensitive resistance to Puccinia hordei-secalini was mapped, suggesting occasional contribution of R genes to nonhost resistance in barley. Quantitative trait loci (QTLs) with effects to multiple rust fungi did not tend to be particularly effective to rust species that were phylogenetically related, as determined from their internal transcribed spacer sequence. We suggest that the QTLs described here play a role as specific and quantitative recognition factors that are specifically negated by the rust to successfully suppress innate immunity.     

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1319-1Abbreviations ff. spp Formae speciales - RIL Recombinant inbred line - DC Double cross - DC-S Progeny produced by selfing of double-cross plants     

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.     

Resistance gene analogs in barley and their relationship to rust resistance genes.

Regions of amino acid conservation in the NBS domain of NBS-LRR resistance proteins facilitated the PCR isolation of eight resistance gene analog (RGA) sequences from genomic DNA of rice, barley, and Aegilops tauschii. These clones and other RGAs previously isolated from maize, rice, and wheat were assigned to 13 classes by DNA-sequence comparison and by their patterns of hybridisation to restricted barley DNA. Using a doubled-haploid mapping population, probes from 12 RGA classes were used to map 17 loci in the barley genome. Many of these probes have been used for mapping in wheat, and the collective data indicate that the positions of orthologous RGAs are conserved between barley and wheat. RGA loci were identified in the vicinity of barley leaf rust resistance loci Rph4, Rph7, and Rph10. Recombinants were identified between RGA loci and Rph7 and Rph10, while a cluster of RGA sequences detected by probe 5.2 cosegregated with Rph4 in 55 F2 lines.     

Golden SusPtrit: a genetically well transformable barley line for studies on the resistance to rust fungi

Key message

We developed ‘Golden SusPtrit’, i.e., a barley line combining SusPtrit’s high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation.

Abstract

Nonhost and partial resistance to Puccinia rust fungi in barley are polygenically inherited. These types of resistance are principally prehaustorial, show high diversity between accessions of the plant species and are genetically associated. To study nonhost and partial resistance, as well as their association, candidate gene(s) for resistance must be cloned and tested in susceptible material where SusPtrit would be the line of choice. Unfortunately, SusPtrit is not amenable to Agrobacterium-mediated transformation. Therefore, a doubled haploid (DH) mapping population (n = 122) was created by crossing SusPtrit with Golden Promise to develop a ‘Golden SusPtrit’, i.e., a barley line combining SusPtrit’s high susceptibility to non-adapted rust fungi with the high amenability of Golden Promise for transformation. We identified nine genomic regions occupied by resistance quantitative trait loci (QTLs) against four non-adapted rust fungi and P. hordei isolate 1.2.1 (Ph.1.2.1). Four DHs were selected for an Agrobacterium-mediated transformation efficiency test. They were among the 12 DH lines most susceptible to the tested non-adapted rust fungi. The most efficiently transformed DH line was SG062N (11–17 transformants per 100 immature embryos). The level of non-adapted rust infection on SG062N is either similar to or higher than the level of infection on SusPtrit. Against Ph.1.2.1, the latency period conferred by SG062N is as short as that conferred by SusPtrit. SG062N, designated ‘Golden SusPtrit’, will be a valuable experimental line that could replace SusPtrit in nonhost and partial resistance studies, especially for stable transformation using candidate genes that may be involved in rust-resistance mechanisms.     

Mapping resistance to powdery mildew in barley reveals a large-effect nonhost resistance QTL

Key message

Resistance factors against non-adapted powdery mildews were mapped in barley. Some QTLs seem effective only to non-adapted mildews, while others also play a role in defense against the adapted form.The durability and effectiveness of nonhost resistance suggests promising practical applications for crop breeding, relying upon elucidation of key aspects of this type of resistance. We investigated which genetic factors determine the nonhost status of barley (Hordeum vulgare L.) to powdery mildews (Blumeria graminis). We set out to verify whether genes involved in nonhost resistance have a wide effectiveness spectrum, and whether nonhost resistance genes confer resistance to the barley adapted powdery mildew. Two barley lines, SusBgt_SC and SusBgt_DC, with some susceptibility to the wheat powdery mildew B. graminis f.sp. tritici (Bgt) were crossed with cv Vada to generate two mapping populations. Each population was assessed for level of infection against four B. graminis ff.spp, and QTL mapping analyses were performed. Our results demonstrate polygenic inheritance for nonhost resistance, with some QTLs effective only to non-adapted mildews, while others play a role against adapted and non-adapted forms. Histology analyses of nonhost interaction show that most penetration attempts are stopped in association with papillae, and also suggest independent layers of defence at haustorium establishment and conidiophore formation. Nonhost resistance of barley to powdery mildew relies mostly on non-hypersensitive mechanisms. A large-effect nonhost resistance QTL mapped to a 1.4 cM interval is suitable for map-based cloning.

     

High-resolution mapping of genes involved in plant stage-specific partial resistance of barley to leaf rust

Partial resistance quantitative trait loci (QTLs) Rphq11 and rphq16 against Puccinia hordei isolate 1.2.1 were previously mapped in seedlings of the mapping populations Steptoe/Morex and Oregon Wolfe Barleys, respectively. In this study, QTL mapping was performed at adult plant stage for the two mapping populations challenged with the same rust isolate. The results suggest that Rphq11 and rphq16 are effective only at seedling stage, and not at adult plant stage. The cloning of several genes responsible for partial resistance of barley to P. hordei will allow elucidation of the molecular basis of this type of plant defence. A map-based cloning approach requires to fine-map the QTL in a narrow genetic window. In this study, Rphq11 and rphq16 were fine-mapped using an approach aiming at speeding up the development of plant material and simplifying its evaluation. The plant materials for fine-mapping were identified from early plant materials developed to produce QTL-NILs. The material was first selected to carry the targeted QTL in heterozygous condition and susceptibility alleles at other resistance QTLs in homozygous condition. This strategy took four to five generations to obtain fixed QTL recombinants (i.e., homozygous resistant at the Rphq11 or rphq16 QTL alleles, homozygous susceptible at the non-targeted QTL alleles). In less than 2 years, Rphq11 was fine-mapped into a 0.2-cM genetic interval and a 1.4-cM genetic interval for rphq16. The strongest candidate gene for Rphq11 is a phospholipid hydroperoxide glutathione peroxidase. Thus far, no candidate gene was identified for rphq16.     

Mapping genes for resistance to barley stripe rust (Puccinia striiformis f. sp. hordei)

Two genes conferring resistance to the barley stripe rust found in Mexico and South America, previously identified as race 24, were mapped to the M arms of barley chromosomes 7 and 4 in a doubled haploid population using molecular markers and the quantitative trait loci (QTL) mapping approach. The resistance gene on chromosome 7 had a major effect, accounting for 57% of the variation in disease severity. The resistance gene on chromosome 4 had a minor effect, accounting for 10% of the variation in trait expression. Two pairs of restriction fragment length polymorphism markers are being used to introgress the resistance genes to North American spring barley using molecular marker-assisted backcrossing.Ore. Agric Exp Stn J no. 10283     

An investigation into the involvement of defense signaling pathways in components of the nonhost resistance of Arabidopsis thaliana to rust fungi also reveals a model system for studying rust fungal compatibility

Seventeen accessions of Arabidopsis thaliana inoculated with the cowpea rust fungus Uromyces vignae exhibited a variety of expressions of nonhost resistance, although infection hypha growth typically ceased before the formation of the first haustorium, except in Ws-0. Compared with wild-type plants, there was no increased fungal growth in ndr1 or eds1 mutants defective in two of the signal cascades regulated by the major class of Arabidopsis host resistance genes. However, in the Col-0 background, infection hyphae of U. vignae and two other rust fungi were longer in sid2 mutants defective in an enzyme that synthesizes salicylic acid (SA), in npr1 mutants deficient in a regulator of the expression of SA-dependent pathogenesis related (PR) genes, and in NahG plants containing a bacterial salicylate hydroxylase. Infection hyphae of U. vignae and U. appendiculatus but not of Puccinia helianthi were also longer in jar1 mutants, which are defective in the jasmonic acid defense signaling pathway. Nevertheless, haustorium formation increased only for the Uromyces spp. and only in sid2 mutants or NahG plants. Rather than the hypersensitive cell death that usually accompanies haustorium formation in nonhost plants, Arabidopsis typically encased haustoria in calloselike material. Growing fungal colonies of both Uromyces spp., indicative of a successful biotrophic relationship between plant and fungus, formed in NahG plants, but only U. vignae formed growing colonies in the sid2 mutants and cycloheximide-treated wild-type plants. Growing colonies did not develop in NahG tobacco or tomato plants. These data suggest that nonhost resistance of Arabidopsis to rust fungi primarily involves the restriction of infection hypha growth as a result of defense gene expression. However, there is a subsequent involvement of SA but not SA-dependent PR genes in preventing the Uromyces spp. from forming the first haustorium and establishing a sufficient biotrophic relationship to support further fungal growth. The U. vignae-Arabidopsis combination could allow the application of the powerful genetic capabilities of this model plant to the study of compatibility as well as nonhost resistance to rust fungi.     

Investigating successive Australian barley breeding populations for stable resistance to leaf rust

Key message

Genome-wide association studies of barley breeding populations identified candidate minor genes for pairing with the adult plant resistance gene Rph20 to provide stable leaf rust resistance across environments.

Abstract

Stable resistance to barley leaf rust (BLR, caused by Puccinia hordei) was evaluated across environments in barley breeding populations (BPs). To identify genomic regions that can be combined with Rph20 to improve adult plant resistance (APR), two BPs genotyped with the Diversity Arrays Technology genotyping-by-sequencing platform (DArT-seq) were examined for reaction to BLR at both seedling and adult growth stages in Australian environments. An integrated consensus map comprising both first- and second-generation DArT platforms was used to integrate QTL information across two additional BPs, providing a total of four interrelated BPs and 15 phenotypic data sets. This enabled identification of key loci underpinning BLR resistance. The APR gene Rph20 was the only active resistance region consistently detected across BPs. Of the QTL identified, RphQ27 on chromosome 6HL was considered the best candidate for pairing with Rph20. RphQ27 did not align or share proximity with known genes and was detected in three of the four BPs. The combination of RphQ27 and Rph20 was of low frequency in the breeding material; however, strong resistance responses were observed for the lines carrying this pairing. This suggests that the candidate minor gene RphQ27 can interact additively with Rph20 to provide stable resistance to BLR across diverse environments.

     

Pyramiding and dissecting disease resistance QTL to barley stripe rust

Quantitative resistance (QR) to disease is usually more durable than qualitative resistance, but its genetic basis is not well understood. We used the barley/barley stripe rust pathosystem as a model for the characterization of the QR phenotype and associated genomic regions. As an intermediate step in the preparation of near-isogenic lines representing individual QTL alleles and combinations of QTL alleles in a homogeneous genetic background, we developed a set of QTL introgression lines in a susceptible background. These intermediate barley near-isogenic (i-BISON) lines represent disease resistance QTL combined in one-, two-, and three-way combinations in a susceptible background. We measured four components of disease resistance on the i-BISON lines: latent period, infection efficiency, lesion size, and pustule density. The greatest differences between the target QTL introgressions and the susceptible controls were for the latter three traits. On average, however, the QTL introgressions also had longer latent periods than the susceptible parent (Baronesse). There were significant differences in the magnitudes of effects of different QTL alleles. The 4H QTL allele had the largest effect, followed by the alleles on 1H and 5H. Pyramiding multiple QTL alleles led to higher levels of resistance in terms of all components of QR except latent period.     

Molecular mapping of a recessive gene for resistance to stripe rust in barley

Barley stripe rust, caused by Puccinia striiformis f. sp. hordei, is one of the most important barley (Hordeum vulgare) diseases in the United States. The disease is best controlled using resistant cultivars. Barley genotype Grannenlose Zweizeilige (GZ) has a recessive gene (rpsGZ) that is effective against all races of P. striiformis f. sp. hordei identified so far in the USA. To develop a molecular map for mapping the gene, F₈ recombinant inbred lines (RILs) were developed from the Steptoe X GZ cross through single-seed descent. Seedlings of the parents and RILs were evaluated for resistance to races PSH-14 and PSH-54 of P. striiformis f. sp. hordei under controlled greenhouse conditions. Genomic DNA was extracted from the parents and 182 F₈ RILs and used for linkage analysis. The resistance gene analog polymorphism (RGAP) technique was used to identify molecular markers for rpsGZ. A linkage group for the gene was constructed with 12 RGAP markers, of which two markers co-segregated with the resistance locus, and two markers were closely linked to the locus with a genetic distance of 0.9 and 2.0 cM, respectively. These four markers were present only in the susceptible parent. The closest marker to the resistance allele was 11.7 cM away. Analyses of two sets of barley chromosome addition lines of wheat with the two RGAP markers that were cosegregating with the susceptibility allele showed that rpsGZ and the markers were located on the long arm of barley chromosome 4H. Further, tests with four simple sequence repeat (SSR) markers confirmed the chromosomal location of the rpsGZ gene and also integrated the RGAP markers into the known SSR-based linkage map of barley. The closest SSR marker EBmac0679 had a genetic distance of 7.5 cM with the gene in the integrated linkage map constructed with the 12 RGAP markers and 4 SSR markers. The information on chromosomal location and molecular markers for rpsGZ should be useful for incorporating this gene into commercial cultivars and combining it with other resistance genes for durable resistance.     

Virulence patterns of barley yellow rust and effective resistance genes to Puccinia striiformis in three parts of Iran

Stripe rust of barley (Hordeum vulgare L.), caused by Puccinia striiformis f. sp. hordei, is a serious problem of barley production in many parts of the world. Monitoring of the pathogen virulence factors and their changes provides basic information for development of an early warning system to breeders and researchers. To monitor the regular virulence changes, trap nurseries comprising of 12 barley differential sets were planted at different parts of Iran in six consecutive years 2007–2012. When the infection and severity under natural infection on susceptible cultivar Afzal as the check was high, then the response of each line was assessed using modified Cobbs scale. Results revealed that no virulence was observed on plants with resistance genes rpsEm1, rpsEm2, rpsHF, Rps4, rpsVa1, rpsVa2 and rpsAst. Therefore, these genes were considered effective genes and can be used to pyramid with those for race-non-specific resistance genes to achieve more durable and highly effective resistance to stripe rust. The plants with the resistance genes rps2, Rps1.b, Rps3 and rpsI5 showed susceptible reaction and virulence was observed on them, thus their resistance genes were considered ineffective.     

The genetics of non-host disease resistance in wheat to barley yellow rust

Non-host resistance is investigated as a potential source of durable resistance. However, the genetics of non-host resistance between closely related plant species and their corresponding pathogens would indicate that in these interactions, non-host resistance primarily involves major genes that operate on a gene-for-gene principal similar to that seen in host resistance. Wheat is a non-host of the barley-attacking form of the fungus responsible for yellow rust, i.e. Puccinia striiformis f. sp. hordei. While P. striiformis f. sp. hordei is generally unable to infect wheat, a partial susceptibility was exhibited by the wheat variety Chinese 166. Consequently, in the cross Lemhi × Chinese 166 two major QTLs for resistance to P. striiformis f. sp. hordei were identified: one on chromosome 1D and a second on 2B. These two QTLs accounted for 43.5% and 33.2% of the phenotypic variance for resistance to barley yellow rust, respectively. In addition, two QTLs of smaller effect were also identified: one on chromosome 5A, contributing 5.1% of the variance and a second on chromosome 6A, contributing 10.9% to the phenotype. The QTL on 6A was derived from the susceptible variety, Chinese 166. In all cases the resistance towards P. striiformis f. sp. hordei was associated with a visual chlorosis/necrosis response typical of race-specific host resistance.     

The rpg4/ Rpg5 stem rust resistance locus in barley; resistance genes and cytoskeleton dynamic

Two closely linked resistance genes, rpg4 and Rpg5, conferring resistance to several races of Puccinia graminis, were cloned and characterized. The Rpg5 gene confers resistance to an isolate of Puccinia graminis f. sp. secalis (Pgs), while rpg4 confers resistance to Puccinia graminis f. sp. tritici (Pgt). Rpg5 is a novel gene containing nucleotide binding site-leucine rich repeat domains in combination with a serine threonine protein kinase domain. High-resolution mapping plus allele and recombinant sequencing identified the rpg4 gene, which encodes an actin depolymerizing factor-like protein (ADF2). Resistance against the Pgt races QCCJ, MCCF, TTKSK (aka Ug99) and RCRS requires both Rpg5 and rpg4, while Rpg5 alone confers resistance to Pgs isolate 92-MN-90. The dependency on the actin modifying protein ADF2 indicates cytoskeleton reorganization or redirection plays a role in pathogen-host interactions. Rpg5 may interact with ADF2 to activate or deactivate its function in the resistance response. Alternatively, Rpg5 could initiate signal transduction leading to resistance in response to detecting ADF2 protein modification. Pgt may redirect the actin cytoskeleton by inducing modifications of ADF2. The redirection of actin could possibly enable the pathogen to develop a haustoria-plant cell cytoskeleton interface for acquisition of nutrients.