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.     

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.     

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.     

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.     

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.     

Leaf rust (Puccinia triticina) resistance in wheat (Triticum aestivum) cultivars grown in Northern Europe 1992-2002

Diversity of resistance to leaf rust caused by Puccinia triticina can be enhanced in wheat (Triticum aestivum) cultivars through a better knowledge of resistance genes that are present in important cultivars and germplasm. Multi-pathotype tests on 84 wheat cultivars grown in Denmark, Finland, Norway and Sweden during 1992-2002 and 39 differential testers enabled the postulation of nine known genes for seedling resistance to leaf rust. Genes Lr1, Lr2a, Lr3, Lr10, Lr13, Lr14a, Lr17, Lr23 and Lr26 were found singly or in combination in 47 of the cultivars (55.9%). The most frequently occurring genes in cultivars grown in Sweden were Lr13 (20.4%), Lr14a (14.8%) and Lr26 (14.8%). Lr14a was the most common gene in cultivars grown in Norway (18.7%), Lr13 in Denmark (35.5%) and Lr10 in Finland (20.0%). Although 28 cultivars (33.3%) exhibited a response pattern that could not be assigned to resistance genes or combinations present in the tester lines, several pathotypes carried virulence and hence these genes or combinations are of limited use. Nine cultivars (10.7%) lacked detectable seedling resistance. One cultivar was resistant to all pathotypes used in the study.     

The interface between virulence and host response to the pathogenic fungus Histoplasma capsulatum

Intracellular pathogens have evolved machinery to evade the immune response in order to survive within a host. Histoplasma capsulatum, one of the intracellular pathogens, is a dimorphic fungus that dodges innate and adaptive immunity; it escapes immunity presumably through virulence factors that permit fungal survival and replication within the host. This review discusses immune factors that contribute to the control of H. capsulatum infection, several host-survival mechanisms H. capsulatum uses, and new techniques that have led to the identification of several H. capsulatum virulence factors, which will most likely aid in the discovery of many more.     

Using chimeric hypoviruses to fine-tune the interaction between a pathogenic fungus and its plant host

Infectious cDNA clones of mild (CHV1-Euro7) and severe (CHV1-EP713) hypovirus strains responsible for virulence attenuation (hypovirulence) of the chestnut blight fungus Cryphonectria parasitica were used to construct viable chimeric viruses. Differences in virus-mediated alterations of fungal colony morphology, growth rate, and canker morphology were mapped to a region of open reading frame B extending from nucleotides 2,363 to 9, 904. By swapping domains within this region, it was possible to generate chimeric hypovirus-infected C. parasitica isolates that exhibited a spectrum of defined colony and canker morphologies. Several severe strain traits were observed to be dominant. It was also possible to uncouple the severe strain traits of small canker size and suppression of asexual sporulation. For example, fungal isolates infected with a chimera containing nucleotides 2363 through 5310 from CHV1-Euro7 in a CHV1-713 background formed small cankers that were similar in size to that caused by CHV1-EP713-infected isolates but with the capacity for producing asexual spores at levels approaching that observed for fungal isolates infected with the mild strain. These results demonstrate that hypoviruses can be engineered to fine-tune the interaction between a pathogenic fungus and its plant host. The identification of specific hypovirus domains that differentially contribute to canker morphology and sporulation levels also provides considerable utility for continuing efforts to enhance biological control potential by balancing hypovirulence and ecological fitness.     

Morphology of the rust fungus Puccinia boroniae revisited

Puccinia boroniae Henns. is a rust fungus endemic to Australia, infecting various Boronia spp. This study describes and illustrates, using light and scanning electron microscopy, the telial stage, teliospore germination and basidiospore production of specimens collected from commercial Boronia plantations in Western Australia. Unusual formation of a single basidiospore per germinating teliospore, and the pycnial stage, observed on Boronia megastigma leaves, are reported for the first time for P. boroniae.     

Activity of trypsin inhibitors in wheat seedlings exposed to pathogenic fungus Tilletia caries and phytohormones

We compared the effect of common bunt (caused by fungus Tilletia caries Tul.) and treatment with phytohormones IAA, ABA, and cytokinins (CK) on the activity of trypsin inhibitors (TI) in wheat seedlings. The experiments were conducted with pathogen-susceptible species of wheat Triticum aestivum L. (cv. Zhnitsa) and resistant species T. timopheevii Zhuk. (accession k-58666 from the collection of All-Russian Institute of Plant Industry). In the resistant wheat, the fungus elevated the activity of TI and the content of CK, whereas in the susceptible wheat, it induced accumulation of IAA. In the seedlings of wheat T. aestivum, TI activity increased under the effect of CK, same as upon the action of pathogen. ABA briefly increased the activity of TI, whereas IAA did not considerably affect it. It was concluded that among the investigated hormones, CK play a leading role in the regulation of defense responses of wheat plants involving TI.