Meinan Wang

Next Generation Sequencing Provides Rapid Access to the Genome of Puccinia striiformis f. sp. tritici, the Causal Agent of Wheat Stripe Rust

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Transcriptome analysis of the wheat–Puccinia striiformis f. sp. tritici interaction

Stripe rust [caused by Puccinia striiformis Westend. f. sp. tritici Eriks. (Pst)] is a destructive disease of wheat (Triticum aestivum L.) worldwide. Genetic resistance is the preferred method for control and the Yr5 gene, originally identified in Triticum spelta var. album, represents a major resistance (R) gene that confers all-stage resistance to all currently known races of Pst in the United States. To identify transcripts associated with the Yr5-mediated incompatible interaction and the yr5-compatible interaction, the Wheat GeneChip was used to profile the changes occurring in wheat isolines that differed for the presence of the Yr5 gene after inoculation with Pst. This time-course study (6, 12, 24 and 48 h post-inoculation) identified 115 transcripts that were induced during the R-gene-mediated incompatible interaction, and 73 induced during the compatible interaction. Fifty-four transcripts were induced in both interactions and were considered as basal defence transcripts, whilst 61 transcripts were specific to the incompatible interaction [hypersensitive response (HR)-specific transcripts] and 19 were specific to the compatible interaction (biotrophic interaction-specific transcripts). The temporal pattern of transcript accumulation showed a peak at 24 h after infection that may reflect haustorial penetration by Pst at ~16 h. An additional 12 constitutive transcript differences were attributed to the presence of Yr5 after eliminating those considered as incomplete isogenicity. Annotation of the induced transcripts revealed that the presence of Yr5 resulted in a rapid and amplified resistance response involving signalling pathways and defence-related transcripts known to occur during R-gene-mediated responses, including protein kinase signalling and the production of reactive oxygen species leading to a hypersensitive response. Basal defence also involved substantial induction of many defence-related transcripts but the lack of R-gene signalling resulted in weaker response.

Construction and characterization of a full-length cDNA library for the wheat stripe rust pathogen (Puccinia striiformis f. sp. tritici).

BackgroundPuccinia striiformis is a plant pathogenic fungus causing stripe rust, one of the most important diseases on cereal crops and grasses worldwide. However, little is know about its genome and genes involved in the biology and pathogenicity of the pathogen. We initiated the functional genomic research of the fungus by constructing a full-length cDNA and determined functions of the first group of genes by sequence comparison of cDNA clones to genes reported in other fungi.ResultsA full-length cDNA library, consisting of 42,240 clones with an average cDNA insert of 1.9 kb, was constructed using urediniospores of race PST-78 of P. striiformis f. sp. tritici. From 196 sequenced cDNA clones, we determined functions of 73 clones (37.2%). In addition, 36 clones (18.4%) had significant homology to hypothetical proteins, 37 clones (18.9%) had some homology to genes in other fungi, and the remaining 50 clones (25.5%) did not produce any hits. From the 73 clones with functions, we identified 51 different genes encoding protein products that are involved in amino acid metabolism, cell defense, cell cycle, cell signaling, cell structure and growth, energy cycle, lipid and nucleotide metabolism, protein modification, ribosomal protein complex, sugar metabolism, transcription factor, transport metabolism, and virulence/infection.ConclusionThe full-length cDNA library is useful in identifying functional genes of P. striiformis.

Yr45, a new wheat gene for stripe rust resistance on the long arm of chromosome 3D

Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most destructive diseases of wheat worldwide. Growing resistant cultivars is the most effective approach to control the disease, but only a few genes confer effective all-stage resistance against the current populations of the pathogen worldwide. It is urgent to identify new genes for diversifying sources of resistance genes and for pyramiding genes for different types of resistance in order to achieve high levels of durable resistance for sustainable control of stripe rust. The common spring wheat genotype ‘PI 181434’, originally from Afghanistan, was resistant in all greenhouse and field tests in our previous studies. To identify the resistance gene(s) PI 181434 was crossed with susceptible genotype ‘Avocet Susceptible’. Adult plants of 103 F2 progeny were tested in the field under the natural infection of P. striiformis f. sp. tritici. Seedlings of the parents, F2 and F3 were tested with races PST-100 and PST-127 of the pathogen under controlled greenhouse conditions. The genetic study showed that PI 181434 has a single dominant gene conferring all-stage resistance. Resistance gene analog polymorphism (RGAP) and simple sequence repeat (SSR) techniques were used to identify molecular markers linked to the gene. A linkage map of 8 RGAP and 2 SSR markers was constructed for the gene using data from the 103 F2 plants and their derived F3 lines tested in the greenhouse. Amplification of the complete set of nulli-tetrasomic lines and selected ditelosomic lines of Chinese Spring with an RGAP marker and the two SSR markers mapped the gene on the long arm of chromosome 3D. Because it is the first gene for stripe rust resistance mapped on chromosome 3DL and different from all previously named Yr genes, the gene in PI 181434 was designated Yr45. Polymorphism rates of the two closest flanking markers, Xwgp115 and Xwgp118, in 45 wheat genotypes were 73.3 and 82.2%, respectively. Single nucleotide polymorphisms (SNPs) were identified in the eight wheat genotypes sharing both flanking markers. The RGAP markers and potential SNP markers should be useful in incorporating the gene into wheat cultivars and in pyramiding it with other genes for durable resistance.

Role of Alternate Hosts in Epidemiology and Pathogen Variation of Cereal Rusts.

Cereal rusts, caused by obligate and biotrophic fungi in the genus Puccinia, are important diseases that threaten world food security. With the recent discovery of alternate hosts for the stripe rust fungus (Puccinia striiformis), all cereal rust fungi are now known to be heteroecious, requiring two distinct plant species serving as primary or alternate hosts to complete their sexual life cycle. The roles of the alternate hosts in disease epidemiology and pathogen variation vary greatly from species to species and from region to region because of different climatic and cropping conditions. We focus this review on rust fungi of small grains, mainly stripe rust, stem rust, leaf rust, and crown rust of wheat, barley, oat, rye, and triticale, with emphases on the contributions of alternate hosts to the development and management of rust diseases.

Barberry as Alternate Host Is Important for Puccinia graminis f. sp. tritici But Not for Puccinia striiformis f. sp. tritici in the U.S. Pacific Northwest

Common barberry (Berberis vulgaris) is the alternate host of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici, under natural conditions in the U.S. Pacific Northwest. Barberry was recently shown to be infected by basidiospores of the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici, under controlled conditions, but it is unclear if barberry plays any role in stripe rust epidemics under natural conditions. Aecial samples of Puccinia spp. collected from barberry plants in the Pacific Northwest from 2010 to 2013 were characterized to species by inoculation on wheat plants under controlled conditions and by molecular markers and sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA. Inoculation of wheat plants with bulked aecia-bearing barberry samples resulted in most P. graminis f. sp. tritici uredia and some P. striiformis f. sp. tritici uredinia. Virulence tests demonstrated that the P. graminis f. sp. tritici isolates were sexually produced, whereas the P. striiformis f. sp. tritici isolates were clonal based on both virulence and simple sequence repeat marker tests, indicating urediniospores from wheat fields landing on barberry leaves as the possible source of P. striiformis f. sp. tritici inoculum. A method for simultaneously testing individual aecia for identifying of P. graminis f. sp. tritici and P. striiformis f. sp. tritici by pathogenicity and ITS markers. Using the method together with ITS sequencing, tested individual aecia were mostly P. graminis f. sp. tritici and occasionally some other formae speciales of P. graminis, but not P. striiformis. The results imply that barberry is essential for stem rust epidemics, but not for stripe rust under the natural conditions in the U.S. Pacific Northwest.

Understanding Molecular Mechanisms of Durable and Non-durable Resistance to Stripe Rust in Wheat Using a Transcriptomics Approach

Stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici, continues to cause severe damage worldwide. Durable resistance is necessary for sustainable control of the disease. High-temperature adult-plant (HTAP) resistance, which expresses when the weather becomes warm and plants grow older, has been demonstrated to be durable. We conducted numerous studies to understand the molecular mechanisms of different types of stripe rust resistance using a transcriptomics approach. Through comparing gene expression patterns with race-specific, all-stage resistance controlled by various genes, we found that a greater diversity of genes is involved in HTAP resistance than in all-stage resistance. The genes involved in HTAP resistance are induced more slowly and their expression induction is less dramatic than genes involved in all-stage resistance. The high diversity of genes and less dramatic induction may explain durability and the incomplete expression level of HTAP resistance. Identification of transcripts may be helpful in identifying resistance controlled by different genes and in selecting better combinations of genes to combine for achieving adequate and durable resistance.

Comparative virulence phenotypes and molecular genotypes of Puccinia striiformis f. sp. tritici, the wheat stripe rust pathogen in China and the United States

Stripe rust (yellow rust) of wheat, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases in both China and the United States. The Chinese and US populations of the stripe rust fungus were compared for their virulence phenotypes on wheat cultivars used to differentiate races of the pathogen in China and the US and molecular genotypes using simple sequence repeat (SSR) markers. From 86 Chinese isolates, 54 races were identified based on reactions on the 17 Chinese differentials and 52 races were identified based on the 20 US differentials. The selected 51 US isolates, representing 50 races based on the US differentials, were identified as 41 races using the Chinese differentials. A total of 132 virulence phenotypes were identified from the 137 isolates based on reactions on both Chinese and US differentials. None of the isolates from the two countries had identical virulence phenotypes on both sets of differentials. From the 137 isolates, SSR markers identified 102 genotypes, of which 71 from China and 31 from the US. The virulence data clustered the 137 isolates into 20 virulence groups (VGs) and the marker data clustered the isolates into seven molecular groups (MGs). Virulence and SSR data had a low (r = 0.34), but significant (P = 0.01) correlation. Principal component analyses using either the virulence data or the SSR data separated the isolates into three groups: group a consisting of only Chinese isolates, group b consisting of both Chinese and US isolates and group c consisting of mostly US isolates. A neighbour-joining tree generated using the molecular data suggested that the P. striiformis f. sp. tritici populations of China and the US in general evolved independently.

Comparative Analysis Highlights Variable Genome Content of Wheat Rusts and Divergence of the Mating Loci

Three members of the Puccinia genus, Puccinia triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

Mapping a Large Number of QTL for Durable Resistance to Stripe Rust in Winter Wheat Druchamp Using SSR and SNP Markers

Winter wheat Druchamp has both high-temperature adult-plant (HTAP) resistance and all-stage resistance to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). The HTAP resistance in Druchamp is durable as the variety has been resistant in adult-plant stage since it was introduced from France to the United States in late 1940s. To map the quantitative trait loci (QTL) for stripe rust resistance, an F8 recombinant inbred line (RIL) population from cross Druchamp × Michigan Amber was phenotyped for stripe rust response in multiple years in fields under natural infection and with selected Pst races under controlled greenhouse conditions, and genotyped with simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers. Composite interval mapping (CIM) identified eight HTAP resistance QTL and three all-stage resistance QTL. Among the eight HTAP resistance QTL, QYrdr.wgp-1BL.2 (explaining 2.36-31.04% variation), QYrdr.wgp-2BL (2.81–15.65%), QYrdr.wgp-5AL (2.27–17.22%) and QYrdr.wgp-5BL.2 (2.42–15.13%) were significant in all tests; and QYrdr.wgp-1BL.1 (1.94–10.19%), QYrdr.wgp-1DS (2.04–27.24%), QYrdr.wgp-3AL (1.78–13.85%) and QYrdr.wgp-6BL.2 (1.69–33.71%) were significant in some of the tests. The three all-stage resistance QTL, QYrdr.wgp-5BL.1 (5.47–36.04%), QYrdr.wgp-5DL (9.27–11.94%) and QYrdr.wgp-6BL.1 (13.07-20.36%), were detected based on reactions in the seedlings tested with certain Pst races. Among the eleven QTL detected in Druchamp, at least three (QYrdr.wgp-5DL for race-specific all-stage resistance and QYrdr.wgp-3AL and QYrdr.wgp-6BL.2 for race non-specific HTAP resistance) are new. All these QTL, especially those for durable HTAP resistance, and their closely linked molecular markers could be useful for developing wheat cultivars with durable resistance to stripe rust.