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.     

Virulence Frequences of Puccinia triticina in Germany and the European Regions of the Russian Federation

From 2001 to 2003, leaf rust was collected in different regions of Germany and the Russian Federation to generate single spore isolates and to study the structure of the pathogen populations by analyses of virulence. The virulence of isolates was tested with 38 near‐isogenic lines each carrying a different resistance gene. The analyses of variance revealed significant effects for the frequency of virulent isolates, the regions and most interactions with years and regions, but no significance was found for the effects of years. In Germany, an increase of virulence frequencies was detected for Lr1 and Lr2a while a decrease was found for Lr3a, Lr3bg and Lr3ka. Such clear trends did not occur in Russia which may be due to the great agroclimatic differences between regions. The variance of the frequency of virulent isolates was used to estimate adequate sample sizes for the analysis of regional populations of leaf rust. This procedure resulted in more reliable information about the dynamic processes within the pathogen populations. In 2002 and 2003, all pathotypes in Germany had a combined virulence to Lr1, Lr2a, Lr2b, Lr15, Lr17 and Lr20 supplemented by a few other genes. The complexity of virulence was lower in the most frequent pathotypes. In Russia virulence to the alleles at locus Lr3 was very common. Using detached leaf segments in Germany and Russia it turned out that the most virulent pathotypes carry 34 and 32 virulence genes, respectively. Virulence to Lr9, Lr19, Lr24 and Lr38 was rare or even absent. The use of major genes, not overcome by corresponding virulent pathotypes, may contribute to more durable types of resistance in case they are combined with genes having different effects, e.g. adult plant resistance.     

Number of genes controlling slow rusting resistance to leaf rust in five spring wheat cultivars

The number of genes controlling slow rusting resistance to leaf rust (Puccinia triticina) was estimated in five spring wheat (Triticum aestivum) cultivars using quantitative formulae. Parents and F₆ families were evaluated in replicated field trials under epidemics initiated by artificial inoculation. The F₆ families resulted from a diallel cross involving the fast-rusting cultivar Yecora 70 and five slow-rusting wheat cultivars: Sonoita 81, Tanager ‘S’, Galvez 87, Ures 81, and Moncho ‘S’. The area under the disease progress curve (AUDPC) was used to measure leaf rust severity over time. Results indicate that cultivar Sonoita 81 has three or four genes, Tanager ‘S’ has two or three genes, Galvez 87 has three genes, and both Ures 81 and Moncho ‘S’ have two genes for slow rusting resistance to leaf rust. Based on this result and previously reported moderate to high narrow-sense heritability estimates for slow rusting resistance in these materials, early-generation selection for slow leaf rusting would be effective.     

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.     

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.     

Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.     

小麦条锈菌II类几丁质合成酶基因PstChsII的克隆及表达特征

摘要:【目的】克隆小麦条锈菌几丁质合成酶基因PstChsII,分析其在小麦条锈菌不同发育时期的表达水平。【方法】利用RT-PCR和PCR技术克隆PstChsII的cDNA序列和基因组序列,利用不同的生物信息学软件对序列进行分析,运用实时荧光定量技术分析基因在孢子、芽管以及不同侵染时间的表达水平。【结果】PstChsII基因（Genbank登录号GQ329851）编码区存在15个内含子,开放阅读框长2727 bp,编码908个氨基酸。PstChsII蛋白C端含有7个跨膜螺旋区,N端含多个保守结构域和“QXR     

小麦隐匿柄锈菌与小麦互作不同阶段的基因差异表达分析

[目的]小麦叶锈病是影响小麦生产主要病害之一,其病原菌新小种的出现和劣势小种上升为优势小种导致抗病品种的抗病性不断被克服.小麦隐匿柄锈菌与小麦互作不同阶段差异表达谱分析对于揭示该病菌致病的分子机制,进而有效防控小麦叶锈病具有重要意义.[方法]利用转录组分析小麦隐匿柄锈菌致病生理小种与感叶锈病小麦品种MuTcLr19亲和...     

28份人工合成小麦对禾谷孢囊线虫、纹枯病、条锈病和叶锈病的抗性

为了发现具有兼抗多种病害的小麦种质,本研究采用田间病圃法和人工接种法,对28份人工合成小麦的禾谷孢囊线虫、纹枯病、条锈病和叶锈病进行了抗性鉴定。人工合成小麦对这些病害表现不同程度的抗性反应。C2和C20对鉴定的4种病害都具有抗性,C5、C10和C25对这些病害都表现感病。8份材料对Heterodera avenae和H. filipjevi两种病原线虫都表现抗性反应型,也有的材料只抗一种线虫。供试材料对纹枯病的抗性表现较好,其中19份材料表现抗性反应型。9份材料对接种的条锈菌小种CY30、CY31、CY32和CY33均表现抗性反应型,5份材料对叶锈菌小种THT和PHT都具抗性。     

Identification of QTLs for partial resistance to leaf rust (Puccinia hordei) in barley

 The partial resistance to leaf rust in barley is a quantitative resistance that is not based on hypersensitivity. To map the quantitative trait loci (QTLs) for partial resistance to leaf rust, we obtained 103 recombinant inbred lines (RILs) by single-seed descent from a cross between the susceptible parent L94 and the partially resistant parent Vada. These RILs were evaluated at the seedling and adult plant stages in the greenhouse for the latent period (LP) of the rust fungus, and in the field for the level of infection, measured as area under the disease progress curve (AUDPC). A dense genetic map based on 561 AFLP markers had been generated previously for this set of RILs. QTLs for partial resistance to leaf rust were mapped using the “Multiple Interval Mapping” method with the putative QTL markers as cofactors. Six QTLs for partial resistance were identified in this population. Three QTLs, Rphq1, Rphq2 and Rphq3, were effective at the seedling stage and contributed approximately 55% to the phenotypic variance. Five QTLs, Rph2, Rphq3, Rphq4, Rphq5, and/or Rphq6 contributed approximtely. 60% of the phenotypic variance and were effective at the adult plant stage. Therefore, only the QTLs Rphq2 and Rhpq3 were not plant-stage dependent. The identified QTLs showed mainly additive effects and only one significant interaction was detected, i.e. between Rphq1 and Rphq2. The map positions of these QTLs did not coincide with those of the race-specific resistance genes, suggesting that genes for partial resistance and genes for hypersensitive resistance represent entirely different gene families. Also, three QTLs for days to heading, of which two were also involved in plant height, were identified in the present recombinant inbred population. These QTLs had been mapped previously on the same positions in different populations. The perspectives of these results for breeding for durable resistance to leaf rust are discussed. Received: 15 July 1997 / Accepted: 30 December 1997     

Effect of gene Lr34 in the enhancement of resistance to leaf rust of wheat

Summary Leaf rust resistance gene Lr34 is present in many wheat cultivars throughout the world that have shown durable resistance to leaf rust. Fourteen pair-wise combinations of Lr34 and seedling leaf rust resistance genes were developed by intercrossing near isogenic Thatcher lines. In both seedling and adult plant tests homozygous paired combinations of specific resistance genes with Lr34 had enhanced resistance relative to either parent to different numbers of isolates that were avirulent to the additional resistance genes. The TcLr34, 18 line also expressed enhanced resistance to specific isolates virulent to Lr18 in seedling and adult plant stages. In rust nursery tests, homozygous lines were more resistant than either parent, if the additional leaf rust gene conditioned an effective of resistance when present singly. The ability of Lr34 to interact with other genes conditioning effective resistance may contribute to the durability of leaf rust resistance in cultivars with Lr34. Contribution 1453 Agriculture Canada     

An N-band marker for gene Lr18 for resistance to leaf rust in wheat

The leaf rust resistance gene, Lr18, of common wheat cultivars has been derived from Triticum timopheevi and is located on chromosome arm 5BL. Chromosome banding (N-banding) analyses revealed that in the wheat cultivars carrying Lr18 that were examined, which had been bred in 6 different countries, chromosome arm 5BL possessed a specific terminal band not carried by their susceptible parental cultivars. It was suggested that this terminal N-band was introduced from T. timopheevi together with Lr18. N-banding analysis of a T. timopheevi strain showed that one of two timopheevi chromosomes had provided Japanese wheat lines containing Lr18 with the terminal band.