Puccinia graminis f. sp. tritici Population Causing Recent Wheat Stem Rust Epidemics in Kazakhstan Is Highly Diverse and Includes Novel Virulence Pathotypes.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a reemerging disease that caused severe epidemics in northern Kazakhstan and western Siberia in the period of 2015 to 2019. We analyzed 51 stem rust samples collected between 2015 and 2017 in five provinces in Kazakhstan. A total of 112 Pgt races were identified from 208 single-pustule isolates. These races are phenotypically and genotypically diverse, and most of them are likely of sexual origin. No differentiation of phenotypes and single-nucleotide polymorphism genotypes was observed between isolates from Akmola and North Kazakhstan provinces, supporting the idea of a wide dispersal of inoculum in the northern regions of the country. Similarities in virulence profiles with Pgt races previously reported in Siberia, Russia, suggest that northern Kazakhstan and western Siberia constitute a single stem rust epidemiological region. In addition to the races of sexual origin, six races reported in Europe, the Caucasus, and East Africa were detected in Kazakhstan, indicating that this epidemiological region is not isolated, and spore inflow from the west occurs. Virulence alone or in combination to several genes effective against the Ug99 race group was detected, including novel virulence on Sr32 + Sr40 and Sr47. The occurrence of a highly diverse Pgt population with virulence to an important group of Sr genes demonstrated the importance of the pathogen's sexual cycle in generating new and potentially damaging virulence combinations.

First Report of Garlic Rust Caused by Puccinia allii on Allium sativum in Minnesota.

High-quality garlic is an emerging crop grown in Minnesota for local markets, community supported agriculture, and select restaurants. In July 2010, Allium sativum cv. German Extra Hardy Porcelain plants showing foliar symptoms typical of rust infection were brought to the Plant Disease Clinic at the University of Minnesota by a commercial grower from Fillmore County, Minnesota. Infected leaves showed circular to oblong lesions (1 to 3 mm long), which ranged in color from yellow-orange (uredinia) to black (telia). Urediniospores collected from uredinia were globoid to ellipsoid, yellowish in color, and measured 18 ± 1 × 30 ± 2 µm with a wall thickness of 2.4 ± 0.5 µm. Teliospores were two celled, 18 ± 3 × 47 ± 10 µm, with a projected cross-sectional area (1) of 826 ± 87 µm2; cell walls were smooth, brown, 1.6 ± 0.3 µm (proximal cell) to 2.1 ± 0.5 µm (distal cell) thick, and 4.2 ± 0.8 µm at the apex. The pathogen was identified as Puccinia allii (2) and a sample was deposited in the U.S. National Fungus Collection (BPI 884132). DNA was extracted from infected leaf tissue and the nuclear ribosomal internal transcribed spacer region (ITS) and 5' end of the large subunit (LS) was amplified and sequenced as described by Anikster et al. (1). The 1,257-bp sequence from the sample collected in Minnesota (GenBank Accession No. JX402206) was identical to ITS/LS sequence of a sample of P. allii collected from garlic in California (GenBank Accession No. AF511077), with the exception that MN sequence contained nine "A"s rather than 10 in the hyper-variable area at the 3' end of the ITS region. P. allii has been shown to be a species complex comprising at least two different types, "leek type" and "garlic type" (1). Based on the ITS sequence and the projected cross-sectional area of the teliospores, the sample of P. allii from MN is consistent with the garlic type. Garlic rust occurred in localized foci late in the growing season and therefore did not cause significant loss to the 2010 crop. Reoccurrence of garlic rust was not reported in either 2011 or 2012 growing seasons in Minnesota. P. allii all but eliminated commercial garlic production in California in the late 1990s (1) and has the potential to cause significant negative impact to the emerging garlic crop in Minnesota. However, the epidemiology of garlic rust in the northern U.S. is not well understood and therefore predicting the risk of the Minnesota garlic crop to rust is difficult. References: (1) Y. Anikster et al. Phytopathology 94:569, 2004. (2) L. J. Szabo et al, Rust. Pages 41-44 in: Compendium of Onion and Garlic Diseases and Pests, Second Edition. H. F. Schwartz and S. K. Mohan, eds. APS Press, St. Paul, 2008.

First Report of Puccinia veronicae-longifoliae on Veronica spicata 'Royal Candles' in Minnesota.

In September 2008, Veronica spicata 'Royal Candles' plants showing foliar symptoms typical of a rust infection were brought to the Plant Disease Clinic at the University of Minnesota. Plants were grown in a local nursery in Dakota County, Minnesota. A dark brown discoloration was apparent on the upper surface of the leaf with lighter brown pustules on the underside. Teliospores collected from the pustules were 2-celled with smooth walls and 36.35 to 48.87 µm long, 11.96 to 18.28 µm wide, and had a wall thickness of 1.33 to 2.61 µm, which is in accordance with type specimen of Puccinia veronicae-longifoliae (4). Pathogen identity was confirmed by comparison of the DNA sequence of nuclear ribosomal RNA region containing the internal transcribed spacer regions 1 and 2, 5.8S and the 5' end of the 28S subunits between herbarium samples from the U.S. National Fungus Collection (BPI 841971/GenBank Accession JQ627617 and BPI 871789/GenBank Accession JQ627618) and the collected specimen (BPI 882886/GenBank Accession JQ627616). P. veronicae-longifoliae was first reported in the United States in 2004 from a commercial nursery in Michigan (2). Veronica rust has also been found in Michigan in 2005 and more recently in 2011 (1). The only other known report of Veronica rust in the United States occurred in Connecticut in 2007 (3). P. veronicae-longifoliae is not considered a quarantine pest by The Animal and Plant Health Inspection Service due to the limited host range, the host not being on the threatened or endangered list and the host being of little economic or environmental importance (2). References: (1) T. A. Dudek et al. MSU Extension News. Retrieved from http://msue.anr.msu.edu/news/veronica_rust_observed_this_season/ , 2011. (2) North American Plant Protection Organization's Phytosanitary Alert System. Retrieved from http://www.pestalert.org/oprDetail.cfm?oprID=129 , 2004. (3) Pundt, L. Floriculture Greenhouse Update. Retrieved from http://www.negreenhouseupdate.info/index.php/july/194-rust-on-veronica , 2007. (4) D. B. O. Savile. Can. J. Bot. 46:631, 1968.

First Report of a New TTKSF Race of Wheat Stem Rust (Puccinia graminis f. sp. tritici) in South Africa and Zimbabwe.

Seven races have been described in the Ug99 race group of Puccinia graminis f. sp. tritici (2). Ug99-related races previously recorded in South Africa are TTKSF, TTKSP, and PTKST (4). In December 2010, severe stem rust infection of the winter wheat cv. Matlabas was observed for the first time in South Africa. Race analysis using the 20 North American (NA) stem rust differential lines and letter code system classified the race as TTKSF. In comparative infection studies in a greenhouse, cv. Matlabas seedlings were susceptible (infection type [IT] 4) to isolate UVPgt61/1 (TTKSF+) collected from Afrikaskop in the eastern Free State, whereas the cultivar was resistant (IT 1 to 2) to stem rust isolates 2013 (TTKSF), UVPgt55 (TTKSF), UVPgt59 (TTKSP), and UVPgt60 (PTKST). Isolate 2013 represents the original collection of race TTKSF in South Africa (1). In addition to the NA differentials, no variation in the IT range of seedlings of lines with Sr7a, 8b, 12, 13, 14, 16, 18, 19, 22, 25, 26, 27, 28, 29, 32, 33, 34, 35, 39, 41, 42, 43, 44, Em, R, Tt2, and Satu was observed between UVPgt61/1 and UVPgt55. With the exception of cv. Matlabas, ITs of 106 South African cultivars likewise did not differentiate UVPgt61/1 and UVPgt55. Seedling IT studies were conducted at least twice. Microsatellite analysis (4) showed that all single pustule isolates established from the original Matlabas isolate formed part of the Ug99 group. When characterized with selected single nucleotide polymorphisms (SNPs), all single pustule isolates shared an identical genotype that differed from UVPgt55 (TTKSF), a foreign introduction into South Africa (1,3). SNP genotype analysis suggests that UVPgt61/1 is genetically dissimilar to UVPgt55, as is Zim1009, another TTKSF+ isolate that was collected from Birchenough in Zimbabwe. Studies are underway to determine the identity of the defeated Sr gene in Matlabas and the cultivar has been added to the South African stem rust differential set. TTKSF+ is the eighth race detected in the Ug99 group. Since no other cultivars or advanced lines were found to carry the Matlabas gene, it is unlikely that race TTKSF+ will threaten wheat production in South Africa. However, the occurrence of a new Ug99-related race emphasizes the variability within this internationally important group. References: (1) W. H. P. Boshoff et al. Plant Dis. 86:922, 2002. (2) R. F. Park et al. Euphytica 179:109, 2011. (3) B. Visser et al. Mol. Plant Pathol. 10:213, 2009. (4) B. Visser et al. Euphytica 179:119, 2011.

Predicting Soybean Rust Incursions into the North American Continental Interior Using Crop Monitoring, Spore Trapping, and Aerobiological Modeling.

Between 2005 and 2009, millions of U.S. and Canadian soybean acres that would have received fungicide application remained untreated for soybean rust due to information disseminated through the Integrated Pest Management Pest Information Platform for Extension and Education (ipmPIPE), increasing North American producers' profits by hundreds of millions of dollars each year. The results of our analysis of Phakopsora pachyrhizi urediniospores in rain collections, aerobiology model output, and observations of soybean rust spread in 2007 and 2008 show a strong correspondence between spore collections and model predictions for the continental interior of North America, where soybean is an important crop. The analysis suggests that control practices based on up-to-date maps of soybean rust observations and associated commentary from Extension Specialists delivered by the ipmPIPE may have suppressed the number and strength of inoculum source areas in the southern states and retarded the northward progress of seasonal soybean rust incursions into continental North America. The analysis further indicates that spore trapping and aerobiological modeling can reduce our reliance on the costly Sentinel Plot Network while maintaining the effectiveness of the ipmPIPE system for soybean rust management.

Identifying and quantifying Phakopsora pachyrhizi spores in rain.

In summers of 2005 and 2006, rain was collected weekly at over 100 selected National Atmospheric Deposition Program/National Trends Network sites across the soybean-growing region of the central and eastern United States. Rain samples were screened for Phakopsora pachyrhizi (causal agent of soybean rust) DNA using a nested real-time polymerase chain reaction assay. Over this time frame, P. pachyrhizi spores were detected in every state in the study, but more frequently in states along the Gulf and Atlantic coasts and along the Ohio River Valley westward to Kansas. A bimodal temporal distribution of samples testing positive for P. pachyrhizi was found in both years. However, there was a greater than threefold increase in the number of samples testing positive for P. pachyrhizi in 2006 compared with 2005, with the most significant increase in August. There was also an increase in the average number of spores per sample in 2006 relative to 2005. Sequence analysis of a subset of positive samples was used to validate the assay results. From the sequence analysis, two reliable polymorphic regions were found, resulting in six distinct genotypes. One genotype was found in 56% of the samples tested, whereas the other genotypes were found less frequently.

Development and characterization of expressed sequence tag-derived microsatellite markers for the wheat stem rust fungus Puccinia graminis f. sp. tritici.

Puccinia graminis f. sp. tritici is the causal agent of stem rust disease in wheat. The rust fungus has caused devastating disease epidemics throughout history and is still posing a potential threat to wheat production in some regions of the world due to the appearance of new races. To develop microsatellite or simple sequence repeat (SSR) markers for use in population genetics studies, a total of 60,579 expressed sequence tag (EST) sequences (reads) generated from P. graminis f. sp. tritici were screened for tandemly repeated di- and tri-nucleotide units using a bioinformatics approach and 708 unisequences containing putative SSR loci with six or more repeat units were identified. Flanking primers were designed for 384 unique SSR loci, which mapped to different locations of the draft genome sequence of the fungus. Of the 384 primer pairs tested, 72 EST-SSR markers were eventually developed, which showed polymorphism among 19 isolates of P. graminis f. sp. tritici and 4 isolates of P. graminis f. sp. secalis evaluated. Thirty-two of the SSR loci were also evaluated in three other rust fungi (P. triticina, P. hordei, and P. coronata f. sp. hordei) for cross-species transferability. These SSR markers derived from ESTs will be useful for characterization of population structures and for gene mapping in P. graminis.

Detection of Virulence to Resistance Gene Sr36 Within the TTKS Race Lineage of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr36 confers a near-immune resistance reaction to many races of Puccinia graminis f. sp. tritici and is highly effective against race TTKSK (syn. Ug99), which possesses unusually broad virulence combinations. Because this gene is widely used in United States soft winter wheat germplasm and cultivars, it has been considered to be an important source of resistance to TTKSK. In 2007, moderately susceptible infection responses were observed on wheat lines and cultivars carrying Sr36 in a field screening nursery for stem rust at Njoro, Kenya. We derived 18 single-pustule isolates from stem rust samples collected from the 2007 Njoro nursery. The isolates were evaluated for virulence on 20 North American stem rust differential lines and on wheat lines and cultivars carrying Sr36, Sr31+Sr36, and Sr24+Sr31. Of the 18 isolates, 10 produced infection types 3+ to 4 on line W2691SrTt-1 (monogenic for Sr36) and other lines that carry Sr36 and belonged to a new virulence phenotype that was not detected in previous years. These isolates were identified as race TTTSK. The remaining eight isolates were identified as races TTKSK (five isolates) and TTKST (three isolates), with avirulence and virulence, respectively, to Sr24. Thirteen simple sequence repeat (SSR) markers were used to examine the genetic relationships among the three races in the TTKS lineage. All isolates in the lineage shared an identical SSR genotype and were clearly different from North American races. In all, 16 wheat cultivars and 60 elite breeding lines, postulated to possess Sr36, were susceptible to race TTTSK. The occurrence of race TTTSK with combined virulence on Sr31 and Sr36 has further broadened the virulence spectrum of the TTKS lineage and rendered an important source of resistance ineffective.

A rapid method for detecting and quantifying bacterial DNA in rust fungal DNA samples.

Bacterial DNA contamination of rust fungal DNA can be a significant problem for sequencing the rust fungus. Sequence assembly is much more difficult if the sequence contigs are mixed with bacterial sequence. A quantitative real-time polymerase chain reaction (qPCR) assay was developed to quantify bacterial DNA within rust fungal DNA samples and the results were compared with those obtained from traditional CFU counts. Real-time PCR showed higher values of DNA contamination than CFU. However, the ranking of samples from low to high for bacterial contamination was consistent between the methods. Reasons for the differences between the methods are discussed. The qPCR assay was tested by adding known quantities of Escherichia coli DNA to Puccinia graminis DNA samples. The assay reliably quantified bacterial contamination at > or = 1.0% of the total sample DNA. When bacterial contamination was <1.0%, fungal DNA also occasionally was amplified, nullifying the quantification measurement. However, primer specificity was not simply the product of the ratio of bacterial DNA to fungal DNA. Bacterial contamination could be quantified below 1.0% if the bacterial DNA concentration was approximately 70 pg/mul or greater. Therefore, spiking the fungal samples with a known concentration of E. coli bacterial DNA successfully eliminated the amplification of fungal DNA, making quantification of contaminating bacterial DNA possible for samples with low contamination levels.

Detection of Virulence to Resistance Gene Sr24 Within Race TTKS of Puccinia graminis f. sp. tritici.

The stem rust resistance gene Sr24 is effective against most races of Puccinia graminis f. sp. tritici, including race TTKS (syn. Ug99), and is used widely in commercial wheat cultivars worldwide. In 2006, susceptible infection responses were observed on wheat lines and cultivars carrying Sr24 in a field stem rust screening nursery at Njoro, Kenya. We derived 28 single-pustule isolates from stem rust samples collected from the 2006 Njoro nursery. The isolates were evaluated for virulence on 16 North American stem rust differential lines; on wheat lines carrying Sr24, Sr31, Sr38, and SrMcN; and on a wheat cultivar with a combination of Sr24 and Sr31. All isolates were identified as race TTKS with additional virulence on Sr31 and Sr38. These isolates were divided into two groups: group A (seven isolates and the two control isolates), producing a low infection type, and group B (21 isolates), producing a high infection type on Sr24, respectively. Isolates of group B represented a new variant of race TTKS with virulence to Sr24. Eighteen simple sequence repeat (SSR) markers were used to examine the genetic relationship between these two groups of isolates in race TTKS and five North American races (MCCF, QCCQ, RCRS, RTHS, and TPMK) that are representative of distinct lineage groups. All isolates of race TTKS shared an identical SSR genotype and were clearly different from North American races. The virulence and SSR data indicated that the new variant of race TTKS with Sr24 virulence likely has arisen via mutation within the TTKS genetic lineage. We propose to revise the North American stem rust nomenclature system by the addition of four genes (Sr24, Sr31, Sr38, and SrMcN) as the fifth set. This revision recognizes the virulence on Sr31 and differentiates isolates within race TTKS into two separate races: TTKSK and TTKST, with avirulence and virulence on Sr24, respectively. The occurrence of race TTKST with combined virulence on Sr24 and Sr31 has substantially increased the vulnerability of wheat to stem rust worldwide.

Detection and identification of four common rust pathogens of cereals and grasses using real-time polymerase chain reaction.

ABSTRACT Puccinia spp. are widespread pathogens of cereals and grasses that annually cause significant yield losses worldwide, especially in barley, oat, and wheat. Urediniospore morphology and early symptom development have limited usefulness for distinguishing Puccinia spp. Therefore, we developed real-time polymerase chain reaction assays for rapid detection of the four rust pathogen species, Puccinia graminis (Pers.:Pers.), P. striiformis (Westend.), P. triticina (Eriks.), and P. recondita (Roberge ex Desmaz.). Duplex assays were constructed for the nuclear rDNA gene, using the variable internal transcribed spacer 1 (ITS1) region to distinguish between species, and the conserved 28S region as an internal control. Species-specific ITS1 primer/probe sets were highly specific and could detect <1 pg of DNA. The species-specific primer/probe sets showed positive results over a linear range of DNA five orders of magnitude or greater. Specificity of the assays was tested using multiple collections representing a range of races and formae speciales within a species. Additionally, assay specificity was evaluated by testing a range of other grass rust pathogens, as well as other fungi. The 28S primer/probe combination was successful in detecting all Puccinia spp. tested within the duplex assays, validating the integrity of each assay. Finally, the assays were used to identify unknown rust fungi infecting pasture grasses.

Morphology, life cycle biology, and DNA sequence analysis of rust fungi on garlic and chives from california.

ABSTRACT In the late 1990s, commercial garlic fields in California (CA) were devastated by an outbreak of rust caused by Puccinia allii. We compared collections of the pathogen from garlic (Allium sativum) and chives (A. schoenoprasum) in central CA and Oregon (OR) to collections from garlic and leek (A. porrum and A. ampeloprasum) in the Middle East. Teliospores from the CA and OR collections were smaller in length, width, and projected cross-sectional area compared with collections from the Middle East. CA and OR collections had a shortened life cycle, in which pycnia and aecia were not formed. Germinating teliospores produced a two-celled promycelium, resulting in two basidiospores, each initially with two nuclei, indicating that this rust was homothallic. In addition, the morphology of the substomatal vesicles was different between the CA-OR (fusiform) and the Middle Eastern (bulbous) collections. DNA sequence analysis of the nuclear ribosomal internal transcribed spacer region showed that the CA and OR rust collections formed a well-supported cluster distinct from the Middle Eastern and European samples. These results suggest that the rust on garlic and chives in CA and OR is a different species than the rust fungus on garlic and leek in the Middle East.

Genetic Variation and Virulence on Lr26 in Puccinia triticina.

ABSTRACT The genetic relationships between isolates of Puccinia triticina virulent on wheat with the Lr26 resistance gene were studied. The diversity within and between isolates of P. triticina from Israel, Europe, and the United States was determined by virulence on near-isogenic Thatcher lines and by random amplified polymorphic DNA. According to the molecular markers, isolates that were virulent on Lr26 had diversity levels similar to those of Lr26 nonpathogenic isolates. Distances between subpopulations of isolates virulent and avirulent on Lr26 varied and were unrelated to the Lr26 virulence phenotype. Cluster analysis suggested four groups, three of which were closely associated with the geographical origin of the isolates-Israel, the United States, and Europe. All four groups included both Lr26 virulent and avirulent pathotypes. The results showed that Lr26 virulent rust pathotypes are as genetically dissimilar as the rest of the population. The cluster analysis showed that the rust population in Israel includes at least two different subpopulations, both of which contain Lr26 virulent and Lr26 avirulent isolates.

Gene Action and Linkage of Avirulence Genes to DNA Markers in the Rust Fungus Puccinia graminis.

ABSTRACT Two strains of the wheat stem rust fungus, Puccinia graminis f. sp. tritici, were crossed on barberry, and a single F(1) progeny strain was selfed. The parents, F(1), and 81 F(2) progeny were examined for virulence phenotypes on wheat differential cultivars carrying stem rust resistance (Sr) genes. For eight Sr differentials, phenotypic ratios are suggestive of single dominant avirulence genes AvrT6, AvrT8a, AvrT9a, AvrT10, AvrT21, AvrT28, AvrT30, and AvrTU. Avirulence on the Sr; (Sr 'fleck') differential showed phenotypic ratios of approximately 15:1, indicating epistatic interaction of two genes dominant for avirulence. Avirulence on Sr9d favored a 3:13 over a 1:3 ratio, possibly indicating two segregating genes-one dominant for avirulence and one dominant for avirulence inhibition. Linkage analysis of eight single dominant avirulence genes and 970 DNA markers identified DNA markers linked to each of these avirulence genes. The closest linkages between AvrT genes and DNA markers were between AvrT6 and the random amplified polymorphic DNA marker crl34-155 (6 centimorgans [cM]) AvrT8a and the amplified fragment length polymorphism marker eAC/mCT-197 (6 cM) and between AvrT9a and the amplified fragment length polymorphism marker eAC/mCT-184 (6 cM). AvrT10 and AvrTU are linked at distance of 9 cM.

Isolation and expression of a host response gene family encoding thaumatin-like proteins in incompatible oat-stem rust fungus interactions.

Four cDNA clones (corresponding to tlp-1, -2, -3, and -4 genes) encoding thaumatin-like (TL), pathogenesis-related proteins were isolated from oat (Avena sativa) infected by an incompatible isolate Pga-1H of the oat stem rust fungus (Puccinia graminis f. sp. avenae). All four cDNA clones contained an open reading frame predicted to encode a 169-amino acid polypeptide with a signal peptide of 21 amino acids at the N-terminus, suggesting that these proteins are transported through a secretory pathway. The amino acid sequences revealed high homology among the four cDNA clones, 80 to 99% identity and 86 to 100% similarity. The tlp genes and several TL protein genes of certain cereals are clustered into a small group that is phylogenetically separate from the major group of TL protein genes of several plant species. In plants infected with the incompatible isolate Pga-1H, or an inappropriate isolate Pgt-8D of P. graminis f. sp. tritici, high levels of tlp gene transcripts accumulated at 42 to 48 h AI and thereafter when hypersensitive host cell death occurred and hyphal growth was inhibited, whereas in plants infected with a compatible isolate Pga-6A, relatively lower amounts of transcripts were detected. Overall, transcript levels were higher with tlp-1 than with the three other genes. Spray with a light mineral oil used as a spore carrier induced transient expression of tlp-1, -2, and -3 genes at 16 to 30 h AI which obscured the initial induction of the tlp genes in response to infection by the pathogens. In contrast, tlp-4 was induced very little by oil spray, so that induction was clearly observed in response to either compatible, incompatible, or inappropriate isolates at 24 to 30 h AI. Wounding leaves by either slicing or puncturing them strongly induced tlp-1 and tlp-3, moderately induced tlp-2, but had no effect on tlp-4. Taken together, the results showed that tlp genes displayed differential responses to oil spray, mechanical wounding, and pathogen infection and that the expression of tlp genes, especially tlp-1, in oat is associated with resistance reactions in response to infection by incompatible and inappropriate isolates of the stem rust fungi.

High-GC primers are useful in RAPD analysis of fungi.

For genetic analysis of fungal DNAs, we have modified the RAPD method to use primers with G + C contents of 80-100%. In RAPD analysis of Puccinia graminis f. sp. tritici DNAs, these primers generated twice the number of both amplification products per primer and polymorphisms among isolates as compared to the standard 60-70% G + C primers. With respect to segregation and genetic similarity, RAPD markers generated by the high-GC primers behaved as do RAPD markers produced by the standard primers. These high-GC primers also yielded increased numbers of amplification products in RAPDs on the DNAs of a broad range of other fungi.

Physical characteristics of the genome of the phytopathogenic fungus Puccinia graminis.

The physical characteristics of the genome of Puccinia graminis f. sp. tritici, the wheat stem rust fungus, were determined by reassociation kinetics. The results indicate that the haploid genome contains 67 Mb and consists of three classes of DNA sequences: (1) 64% unique; (2) 30% repetitive; and (3) 4% foldback. The repetitive sequences have a total complexity of 390 kb and are repeated an average of 52 times. The base composition was 45.3% G+C based on an analysis of the DNA melting temperature. The average amount of DNA per ungerminated urediniospore by diphenylamine assay, corrected for losses during extraction, was 435 fg. This was three times the expected value (147 fg) for dikaryotic spores with nuclei in the G1 phase of the cell cycle, an indication that the spores were in G2.

Molecular cloning and analysis of abundant and stage-specific mRNAs from Puccinia graminis.

To characterize highly expressed mRNAs from germinated urediniospores of Puccinia graminis f. sp. tritici, we isolated 68 cDNA clones of abundant mRNA species belonging to at least six homology groups. The two most abundant homology groups, HG1 and HG2, contained 54 of the 68 cDNA clones and accounted for 2.4 and 0.6% of the poly(A)+ RNA in germinated urediniospores, respectively. By sampling different developmental stages of the uredinial cycle, we showed that the uam transcript, corresponding to HG2, accumulated in all stages of hyphal and urediniospore development, whereas the accumulation of usp transcript, corresponding to HG1, was specific to the sporulation stage. Southern blot analysis indicated that usp is a small gene family consisting of three to four members. Sequence analysis of 10 cDNA clones indicated that two different members of the usp gene family were expressed in germinated urediniospores. This gene family encodes small hydrophobic polypeptides of 113 amino acids with an unusual amino acid composition, in that alanine, glycine, leucine, and proline represent 48% of the protein. These polypeptides are predicted to be localized extracellular because they contain a putative signal sequence and may be functionally related to hydrophobins, a family of small hydrophobic proteins abundantly expressed during sporulation in Schizophyllum commune and Aspergillus nidulans. The uam and usp genes deserve further investigation, including isolation of genomic clones. The regulatory regions of the uam gene, which is highly expressed in hyphae, may be useful in the construction of a transformation vector for rust fungi.

The transit peptide of a chloroplast thylakoid membrane protein is functionally equivalent to a stromal-targeting sequence.

The role of transit peptides in intraorganellar targeting has been studied for a chlorophyll a/b binding (CAB) polypeptide of photosystem II (PSII) and the small subunit of ribulose-1,5-bisphosphate carboxylase (RBCS) from Pisum sativum (pea). These studies have involved in vitro import of fusion proteins into isolated pea chloroplasts. Fusion of the CAB transit peptide to RBCS mediates import to the stroma, as evidenced by assembly of RBCS with chloroplast-synthesized large subunit (RBCL) to form holoenzyme. Similarly, fusion of the RBCS transit peptide to the mature CAB polypeptide mediates import and results in integration of the processed CAB protein into the thylakoid membrane. Correct integration was indicated by association with PSII and assembly with chlorophyll to form the light-harvesting chlorophyll a/b protein complex (LHCII). We interpret these results as evidence that the CAB transit peptide is functionally equivalent to a stromal-targeting sequence and that intraorganellar sorting of the CAB protein must be determined by sequences residing within the mature protein. Our results and those of others suggest that import and integration of CAB polypeptides into the thylakoid proceeds via the stroma.