Population divergence in the wheat leaf rust fungus Puccinia triticina is correlated with wheat evolution

Co-evolution of fungal pathogens with their host species during the domestication of modern crop varieties has likely affected the current genetic divergence of pathogen populations. The objective of this study was to determine if the evolutionary history of the obligate rust pathogen on wheat, Puccinia triticina, is correlated with adaptation to hosts with different ploidy levels. Sequence data from 15 loci with different levels of polymorphism were generated. Phylogenetic analyses (parsimony, Bayesian, maximum likelihood) showed the clear initial divergence of P. triticina isolates collected from Aegilops speltoides (the likely B genome donor of modern wheat) in Israel from the other isolates that were collected from tetraploid (AB genomes) durum wheat and hexaploid (ABD genomes) common wheat. Coalescence-based genealogy samplers also indicated that P. triticina on A. speltoides, diverged initially, followed by P. triticina isolates from durum wheat in Ethiopia and then by isolates from common wheat. Isolates of P. triticina found worldwide on cultivated durum wheat were the most recently coalesced and formed a clade nested within the isolates from common wheat. By a relative time scale, the divergence of P. triticinia as delimited by host specificity appears very recent. Significant reciprocal gene flow between isolates from common wheat and isolates from durum wheat that are found worldwide was detected, in addition to gene flow from isolates on common wheat to isolates on durum wheat in Ethiopia.     

Development of simple sequence repeat markers for the plant pathogenic rust fungus Puccinia triticina

Eighteen polymorphic di‐ and trinucleotide simple sequence repeat markers were developed for the phytopathogenic rust fungus Puccinia triticina. The allelic diversity varied from two to nine alleles per locus. Levels of observed heterozygosity ranged from 0.095 to 0.952. Seven of the loci deviated significantly from Hardy–Weinberg equilibrium (P < 0.002) with 70% having levels of observed heterozygosity higher than expected heterozygosity. Null allele(s) were observed for locus PtSSR76 with a frequency of 9%. A preliminary screen of other cereal rust fungi (P. coronata, P. graminis, P. recondita and P. striiformis) indicated that these primer pairs are specific to P. triticina.     

The role of reactive oxygen species in the virulence of wheat leaf rust fungus Puccinia triticina

Reactive oxygen species (ROS) play an important role during host–pathogen interactions and are often an indication of induced host defence responses. In this study, we demonstrate for the first time that Puccinia triticina (Pt) generates ROS, including superoxide, H₂O₂ and hydroxyl radicals, during wheat infection. Through pharmacological inhibition, we found that ROS are critical for both Pt urediniospore germination and pathogenic development on wheat. A comparative RNA-Seq analysis of different stages of Pt infection process revealed 291 putative Pt genes associated with the oxidation–reduction process. Thirty-seven of these genes encode known proteins. The expressions of five Pt genes, including PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod, were subsequently verified using RT-qPCR analysis. The results show that the expressions of PtNoxA, PtNoxB, PtNoxR, PtCat and PtSod are up-regulated during urediniospore germination. In comparison, the expressions of PtNoxA, PtNoxB, PtNoxR and PtCat are down-regulated during wheat infection from 12 to 120 h after inoculation (HAI), whereas the expression of PtSod is up-regulated with a peak of expression at 120 HAI. We conclude that ROS are critical for the full virulence of Pt and a coordinate down-regulation of PtNox genes may be important for successful infection in wheat.     

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.     

Mapping of QTLs prolonging the latent period of Puccinia triticina infection in wheat

Slow rusting is considered a crucial component of durable resistance to wheat leaf rust caused by Puccinia triticina and is often expressed in the form of a prolonged latent period. Selection for a longer latent period is considered an effective approach to developing wheat cultivars with improved durable resistance to leaf rust. A recombinant inbred line (RIL) population derived from CI 13227 (long latent period) × Suwon 92 (short latent period) was phenotyped for latent period in two greenhouse experiments in separate years, and amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) markers were analyzed in the same population. Among the RILs, the frequency distribution for latent period was continuous, and latent period was highly correlated between years (r=0.94, P<0.0001). A quantitative trait locus (QTL) prolonging the latent period of P. triticina, designated as QLrlp.osu-2DS, explained 42.8% and 54.5% of the phenotypic and genetic variance in the two experiments, respectively. QLrlp.osu-2DS was mapped on the distal region of chromosome 2DS. Two other QTLs for latent period, QLrlp.osu-2B and QLrlp.osu-7BL, were localized on chromosome 2B and the long arm of chromosome 7B, respectively. Multiple regression analysis showed that these three QTLs collectively explained 58.0% and 73.8% of the phenotypic and genetic variance over two experiments, respectively. Fourteen RILs that carried all three alleles for long latent period at three AFLP loci flanking QLrlp.osu-2DS, QLrlp.osu-2B, and QLrlp.osu-7BL had a mean latent period of 12.5 days, whereas 13 RILs without any long-latent-period alleles at the corresponding loci had a mean latent period of 7.4 days. Three SSR markers closely linked to these QTLs have potential to be applied in marker-assisted selection for prolonged latent period in wheat.     

Isolation of 12 microsatellite loci,using an enrichment protocol,in the phytopathogenic fungus Puccinia triticina

We report the characterization of 12 polymorphic microsatellite markers in the biotrophic fungus Puccinia triticina, the causal agent of leaf rust on wheat. An enrichment protocol was used to isolate microsatellite loci and polymorphism was explored with 15 European isolates. Significant level of cross‐amplification (44% of the loci) was found in P. striiformis.     

Characterisation and mapping of gene Lr73 conferring seedling resistance to Puccinia triticina in common wheat

Key message

A gene conferring seedling resistance to Puccinia triticina was mapped to chromosome 2BS in the wheat Morocco. The gene was shown to be distinct and was therefore designated Lr73.

Abstract

The wheat genotype Morocco, widely susceptible to isolates of Puccinia triticina, was resistant to an Australian isolate of this pathogen collected in 2004. Genetic studies established that the resistance in Morocco was also present the Australian wheat genotypes Avocet, Halberd, Harrier, Tincurrin and a selection of cultivar Warigal lacking the resistance gene Lr20. Genetic studies based on a cross with Halberd showed that the gene is dominant and located on chromosome 2BS (XwPt8760—4 cM—Lr73—1.4 cM—XwPt8235). The gene was genetically independent of the Lr13, Lr16 and Lr23 loci, also located on chromosome 2BS, indicating that it is distinct. The locus designation Lr73 was therefore assigned. On the basis of multi-pathotype tests, it is likely Lr73 is also present in the Australian wheat cultivars Clearfield STL, Federation (with Lr10), Gatcher (with Lr10 and Lr27+Lr31), Marombi (with Lr1 and Lr37), Pugsley (with Lr1 and Lr37), Spear (with Lr1), Stiletto and Tarsa (with Lr1). Gene Lr73 is unlikely to be of value in resistance breeding. However, recognising Lr73 is important to avoid its inadvertent selection in breeding programmes. Furthermore, the apparent rarity of avirulence for genes like Lr73, sometimes referred to as “fossil” resistance genes, makes them of interest in terms of the evolution of disease resistance in host plants and of virulence in the respective rust pathogens.     

Analysis of the wheat and Puccinia triticina (leaf rust) proteomes during a susceptible host-pathogen interaction

Wheat leaf rust is caused by the fungus Puccinia triticina. The genetics of resistance follows the gene-for-gene hypothesis, and thus the presence or absence of a single host resistance gene renders a plant resistant or susceptible to a leaf rust race bearing the corresponding avirulence gene. To investigate some of the changes in the proteomes of both host and pathogen during disease development, a susceptible line of wheat infected with a virulent race of leaf rust were compared to mock-inoculated wheat using 2-DE (with IEF pH 4-8) and MS. Up-regulated protein spots were excised and analyzed by MALDI-QqTOF MS/MS, followed by cross-species protein identification. Where possible MS/MS spectra were matched to homologous proteins in the NCBI database or to fungal ESTs encoding putative proteins. Searching was done using the MASCOT search engine. Remaining unmatched spectra were then sequenced de novo and queried against the NCBInr database using the BLAST and MS BLAST tools. A total of 32 consistently up-regulated proteins were examined from the gels representing the 9-day post-infection proteome in susceptible plants. Of these 7 are host proteins, 22 are fungal proteins of known or hypothetical function and 3 are unknown proteins of putative fungal origin.     

Characteristic expression of wheat PR5 gene in response to infection by the leaf rust pathogen,Puccinia triticina

TaLr35PR5 gene was obtained from the gDNA and cDNA of TcLr35 wheat. It was induced by Puccinia triticina, ABA and SA, but TaLr35PR5 was induced earlier and its expression level was higher in the incompatible interaction than that in the compatible interaction. In addition, the accumulations of TaLr35PR5 increased stably and showed significant peak challenged by P. triticina at different growth and development periods of TcLr35 wheat while it maintained similar level and changed little in mock inoculated. Western blottings were conducted to confirm that TaLr35PR5 be induced by P. triticina infection at the protein expression level. Similar to the expression patterns of TaLr35PR5 at RNA levels, the accumulations of TaLr35PR5 protein were weak in the seedling stage, then increased to the peak and kept constant levels at the mature stage which is consistent with the expression feature of Lr35 gene as an adult plant resistance gene.     

Transient expression and insertional mutagenesis of Puccinia triticina using biolistics

The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-α, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.     

Rapid speciation following recent host shifts in the plant pathogenic fungus Rhynchosporium

Agriculture played a significant role in increasing the number of pathogen species and in expanding their geographic range during the last 10,000 years. We tested the hypothesis that a fungal pathogen of cereals and grasses emerged at the time of domestication of cereals in the Fertile Crescent and subsequently speciated after adaptation to its hosts. Rhynchosporium secalis, originally described from rye, causes an important disease on barley called scald, although it also infects other species of Hordeum and Agropyron. Phylogenetic analyses based on four DNA sequence loci identified three host-associated lineages that were confirmed by cross-pathogenicity tests. Bayesian analyses of divergence time suggested that the three lineages emerged between approximately 1200 to 3600 years before present (B.P.) with a 95% highest posterior density ranging from 100 to 12,000 years B.P. depending on the implemented clock models. The coalescent inference of demographic history revealed a very recent population expansion for all three pathogens. We propose that Rhynchosporium on barley, rye, and Agropyron host species represent three cryptic pathogen species that underwent independent evolution and ecological divergence by host-specialization. We postulate that the recent emergence of these pathogens followed host shifts. The subsequent population expansions followed the expansion of the cultivated host populations and accompanying expansion of the weedy Agropyron spp. found in fields of cultivated cereals. Hence, agriculture played a major role in the emergence of the scald diseases, the adaptation of the pathogens to new hosts and their worldwide dissemination.     

Development of simple sequence repeat markers for the plant pathogenic rust fungus,Puccinia graminis

Twenty‐four dinucleotide simple sequence repeat markers were developed for the phytopathogenic fungus, Puccinia graminis. The identified loci were polymorphic, with allelic diversity ranging from two to 11 alleles. Observed and expected levels of heterozygosity ranged from 0.000 to 0.960 and from 0.113 to 0.846, respectively. Fourteen of the loci deviated significantly from Hardy–Weinberg equilibrium. Null alleles were observed for 10 of the 24 loci with a frequency of 4–16%. A preliminary screen of other Puccinia cereal rust fungi (P. coronata, P. striiformis and P. triticina) indicated that these primer pairs are specific to P. graminis.     

Pathogenic Specialization of Puccinia triticina in Andalusia from 1998 to 2000

Wheat leaf rust (Puccinia triticina) is becoming a serious concern in Spanish wheat, especially on durum wheat where acreage has enormously increased. Host resistance is the preferred method of disease control, but the virulence spectrum of the leaf rust population in Spain is currently unknown. In order to deploy effective Lr genes, this study was conducted to characterize the virulence spectrum of leaf rust in Andalusia (Spain). Isolates were obtained from surveys of wheat fields across Andalusia from 1998 to 2000. From 56 isolates phenotyped, 35 pathotypes were identified. Virulence to Lr10, Lr11, Lr14a, Lr14b and Lr18 was high (>96%), while virulence to Lr9 and Lr24 were not found. None of the isolates collected from durum wheat were virulent to Lr1, Lr3, Lr3ka, Lr3bg, Lr15, Lr16 and Lr17, while many of the isolates collected on bread wheat showed virulence on these genes, indicating a certain specialization in the leaf rust infecting durum wheat. Population dynamics of current wheat leaf rust pathotypes in terms of mutation and migration are discussed.     

Identification and molecular tagging of a gene from PI 289824 conferring resistance to leaf rust (Puccinia triticina) in wheat

Host-plant resistance is the most economically viable and environmentally responsible method of control for Puccinia triticina, the causal agent of leaf rust in wheat (Triticum aestivum L.). The identification and utilization of new resistance sources is critical to the continued development of improved cultivars as shifts in pathogen races cause the effectiveness of widely deployed genes to be short lived. The objectives of this research were to identify and tag new leaf rust resistance genes. Forty landraces from Afghanistan and Iran were obtained from the National Plant Germplasm System and evaluated under field conditions at two locations in Texas. PI 289824, a landrace from Iran, was highly resistant under field infection. Further evaluation revealed that PI 289824 is highly resistant to a broad spectrum of leaf rust races, including the currently prevalent races of leaf rust in the Great Plains area of the USA. Eight F₁ plants, 176 F₂ individuals and 139 F_2:3 families of a cross between PI 289824 and T112 (susceptible) were evaluated for resistance to leaf rust at the seedling stage. Genetic analysis indicated resistance in PI 289824 is controlled by a single dominant gene. The AFLP analyses resulted in the identification of a marker (P39 M48-367) linked to resistance. The diagnostic AFLP band was sequenced and that sequence information was used to develop an STS marker (TXW₂₀₀) linked to the gene at a distance of 2.3 cM. The addition of microsatellite markers allowed the gene to be mapped to the short arm of Chromosome 5B. The only resistance gene to be assigned to Chr 5BS is Lr52. The Lr52 gene was reported to be 16.5 cM distal to Xgwm443 while the gene in PI 289824 mapped 16.7 cM proximal to Xgwm443. Allelism tests are needed to determine the relationship between the gene in PI 289824 and Lr52. If the reported map positions are correct, the gene in PI 289824 is unique.