Stem Rust Fungus Puccinia Graminis Research Articles

Nonhost Resistance of Thinopyrum ponticum to Puccinia graminis f. sp. tritici and the Effects of the Sr24, Sr25, and Sr26 Genes Introgressed to Wheat

The damage to wheat crops by stem rust poses a threat to the food security of the world’s population. The species Thinopyrum ponticum (Podpěra, 1902) (Z.-W. Liu and R.-C. Wang, 1993) is a non-host for the stem rust fungus Puccinia graminis f. sp. tritici Eriks. and Henn. (Pgt). The Sr24, Sr25, and Sr26 genes, transferred from the Th. ponticum to the wheat gene pool, protect cultivars from the disease in different regions of the world. The study of the non-host resistance (NHR) of Th. ponticum and the effects of the introgressed Sr24, Sr25, and Sr26 genes in wheat is important for breeding cultivars with durable resistance to stem rust. The aim of the research is to study the interaction of Pgt with Th. ponticum and common wheat lines with the Sr24, Sr25, and Sr26 genes, in addition to determining the role of ROS in resistance. Wheat lines with Sr24, Sr25, and Sr26 were resistant to the West Siberian Pgt population. Using cytological methods, it was found that the NHR of Th. ponticum and Sr24, Sr25, and Sr26 led to inhibition of the most inoculumdevelopment on the plant surface. This was mainly due to the suppression of the appressoria development and their death at the stage of penetration into the stomata. Upon contact of Pgt appressoria with stomatal guard cells, the generation of the superoxide anion O2•− was revealed. This interaction is similar to the stomatal immunity of Arabidopsis thaliana to non-pathogenic bacteria. The results of our studies show that the Sr24, Sr25, and Sr26 genes reproduce the action of some NHR mechanisms in wheat.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation

BackgroundSilencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive.ResultsWe use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5′ uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5′ adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes.ConclusionsWe conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Registration of hard white winter wheat germplasms KS14U6380R5, KS16U6380R10, and KS16U6380R11 with adult plant resistance to stem rust

AbstractResistance to the Ug99 group of races of the stem rust fungus Puccinia graminis f. sp. tritici is limited in winter wheat (Triticum aestivum L.) germplasm adapted to the Great Plains of the United States. Our objective was to generate regionally adapted hard winter wheat germplasm with combinations of adult plant resistance genes that are expected to provide durable resistance. KS14U6380R5 (Reg. no. GP‐1043, PI 689115), KS16U6380R10 (Reg. no. GP‐1044, PI 689116), and KS16U6380R11 (Reg. no. GP‐1045, PI 689117) were derived from backcrosses of the hard white winter wheat germplasm KS05HW14 to the stem rust‐resistant Kenyan spring wheat cultivar ‘Kingbird’. KS14U6380R5, KS16U6380R11, and KS16U6380R10 were developed by pedigree selection and were initially evaluated as U6380‐11‐2R‐0A, U6380‐210‐2R‐0A, and U6380‐148‐4R‐2T, respectively. The germplasms were developed by the USDA‐ARS and jointly released with the Kansas State University Agricultural Experiment Station. These germplasms provide parents for development of hard winter wheat cultivars with durable resistance to stem rust.

Identification and Validation of a Common Stem Rust Resistance Locus in Two Bi-parental Populations.

Races belonging to Ug99 lineage of stem rust fungus Puccinia graminis f. sp. tritici (Pgt) continue to pose a threat to wheat (Triticum aestivum L.) production in various African countries. Growing resistant varieties is the most economical and environmentally friendly control measure. Recombinant inbred line (RIL) populations from the crosses of susceptible parent ‘Cacuke’ with the resistant parents ‘Huhwa’ and ‘Yaye’ were phenotyped for resistance at the seedling stage to Pgt race TTKSK (Ug99) and in adult plants in field trials at Njoro, Kenya for two seasons in 2016. Using the Affymetrix Axiom breeders SNP array, two stem rust resistance genes, temporarily designated as SrH and SrY, were identified and mapped on chromosome arm 2BL through selective genotyping and bulked segregant analysis (BSA), respectively. Kompetitive allele specific polymorphism (KASP) markers and simple sequence repeat (SSR) markers were used to saturate chromosome arm 2BL in both RIL populations. SrH mapped between markers cim109 and cim114 at a distance of 0.9 cM proximal, and cim117 at 2.9 cM distal. SrY was flanked by markers cim109 and cim116 at 0.8 cM proximal, and IWB45932 at 1.9 cM distal. Two Ug99-effective stem rust resistance genes derived from bread wheat, Sr9h and Sr28, have been reported on chromosome arm 2BL. Infection types and map position in Huhwa and Yaye indicated that Sr28 was absent in both the parents. However, susceptible reactions produced by resistant lines from both populations against Sr9h-virulent race TTKSF+ confirmed the presence of a common resistance locus Sr9h in both lines. Test of allelism is required to establish genetic relationships between genes identified in present study and Sr9h. Marker cim117 linked to SrH was genotyped on set of wheat lines with Huhwa in the pedigree and is advised to be used for marker assisted selection for this gene, however, a combination of phenotypic and genotypic assays is desirable for both genes especially for selection of Sr9h in breeding programs.

Genomic analyses and expression evaluation of thaumatin-like gene family in the cacao fungal pathogen Moniliophthora perniciosa

Thaumatin-like proteins (TLPs) are found in diverse eukaryotes. Plant TLPs, known as Pathogenicity Related Protein (PR-5), are considered fungal inhibitors. However, genes encoding TLPs are frequently found in fungal genomes. In this work, we have identified that Moniliophthora perniciosa, a basidiomycete pathogen that causes the Witches' Broom Disease (WBD) of cacao, presents thirteen putative TLPs from which four are expressed during WBD progression. One of them is similar to small TLPs, which are present in phytopathogenic basidiomycete, such as wheat stem rust fungus Puccinia graminis. Fungi genomes annotation and phylogenetic data revealed a larger number of TLPs in basidiomycetes when comparing with ascomycetes, suggesting that these proteins could be involved in specific traits of mushroom-forming species. Based on the present data, we discuss the contribution of TLPs in the combat against fungal competitors and hypothesize a role of these proteins in M. perniciosa pathogenicity.

Determination of the Role of Berberis spp. in Wheat Stem Rust in China.

Previous studies on the relationship of barberry (Berberis spp.) and wheat stem rust suggested that, although some barberry species can serve as alternate hosts for the stem rust fungus Puccinia graminis f. sp. tritici, barberry plants play no role in wheat stem rust development and virulence variation of P. graminis f. sp. tritici in China. In the present study, severe rust infections on Berberis shensiana, B. brachypoda, B. potaninii, B. soulieana, and B. aggregata were observed during field surveys in 2011 and 2012. Through artificial inoculation of wheat seedlings ('Mingxian 169') under greenhouse conditions with aeciospores from naturally infected barberry plants, 185 isolates of P. graminis f. sp. tritici were obtained. From the 27 selected isolates that were tested on a set of wheat genotypes used to differentiate P. graminis f. sp. tritici races in China, 18 races were identified, of which 8 races were new and others were of Chinese 21 and 34 race groups. In addition to the information of virulence or avirulence patterns on the Chinese differentials, none of the races were virulent to resistance gene Sr31. The virulence frequencies based on individual Sr genes or differentials ranged from 0 to 96%. Using molecular markers for P. graminis f. sp. tritici, 75 of 4,036 samples of single aecia were identified as P. graminis f. sp. tritici. The low detectable rate of P. graminis f. sp. tritici was verified based on both infection and molecular marker tests. This observation may be correlated with the low levels of wheat stem rust in the surveyed regions. This study shows that P. graminis f. sp. tritici can produce new races through sexual reproduction on Berberis spp. in China.

Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin. The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ∼10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates. Some of these mutations could explain their novel virulence phenotypes.

A Bacterial Type III Secretion Assay for Delivery of Fungal Effector Proteins into Wheat

Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.

Characterization of a Tryptophan 2-Monooxygenase Gene from Puccinia graminis f. sp. tritici Involved in Auxin Biosynthesis and Rust Pathogenicity

The plant hormone indole-3-acetic acid (IAA) is best known as a regulator of plant growth and development but its production can also affect plant-microbe interactions. Microorganisms, including numerous plant-associated bacteria and several fungi, are also capable of producing IAA. The stem rust fungus Puccinia graminis f. sp. tritici induced wheat plants to accumulate auxin in infected leaf tissue. A gene (Pgt-IaaM) encoding a putative tryptophan 2-monooxygenase, which makes the auxin precursor indole-3-acetamide (IAM), was identified in the P. graminis f. sp. tritici genome and found to be expressed in haustoria cells in infected plant tissue. Transient silencing of the gene in infected wheat plants indicated that it was required for full pathogenicity. Expression of Pgt-IaaM in Arabidopsis caused a typical auxin expression phenotype and promoted susceptibility to the bacterial pathogen Pseudomonas syringae pv. tomato DC3000.

Exploiting regulatory variation to identify genes underlying quantitative resistance to the wheat stem rust pathogen Puccinia graminis f. sp. tritici in barley

We previously mapped mRNA transcript abundance traits (expression-QTL or eQTL) using the Barley1 Affymetrix array and 'whole plant' tissue from 139 progeny of the Steptoe x Morex (St/Mx) reference barley mapping population. Of the 22,840 probesets (genes) on the array, 15,987 reported transcript abundance signals that were suitable for eQTL analysis, and this revealed a genome-wide distribution of 23,738 significant eQTLs. Here we have explored the potential of using these mRNA abundance eQTL traits as surrogates for the identification of candidate genes underlying the interaction between barley and the wheat stem rust fungus Puccinia graminis f. sp. tritici. We re-analysed quantitative 'resistance phenotype' data collected on this population in 1990/1991 and identified six loci associated with barley's reaction to stem rust. One of these coincided with the major stem rust resistance locus Rpg1, that we had previously positionally cloned using this population. Correlation analysis between phenotype values for rust infection and mRNA abundance values reported by the 22,840 GeneChip probe sets placed Rpg1, which is on the Barley1 GeneChip, in the top five candidate genes for the major QTL on chromosome 7H corresponding to the location of Rpg1. A second co-located with the rpg4/Rpg5 stem rust resistance locus that has been mapped in a different population and the remaining four were novel. Correlation analyses identified candidate genes for the rpg4/Rpg5 locus on chromosome 5H. By combining our data with additional published mRNA profiling data sets, we identify a putative sensory transduction histidine kinase as a strong candidate for a novel resistance locus on chromosome 2H and compile candidate gene lists for the other three loci.

Parallel expression profiling of barley–stem rust interactions

The dominant barley stem rust resistance gene Rpg1 confers resistance to many but not all pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici (Pgt). Transformation of Rpg1 into susceptible cultivar Golden Promise rendered the transgenic plants resistant to Pgt pathotype MCC but not to Pgt pathotype QCC. Our objective was to identify genes that are induced/repressed during the early stages of pathogen infection to elucidate the molecular mechanisms and role of Rpg1 in defense. A messenger ribonucleic acid expression analysis using the 22K Barley1 GeneChip was conducted in all pair-wise combinations of two isolines (cv. Golden Promise and Rpg1 transgenic line G02-448F-3R) and two Pgt pathotypes (MCC and QCC) across six time points. Analysis showed that a total of 34 probe sets exhibited expression pattern differences between Golden Promise (susceptible) and G02-448F-3R (resistant) infected with Pgt-MCC. A total of 14 probe sets exhibited expression pattern differences between Pgt-MCC (avirulent) and Pgt-QCC (virulent) inoculated onto G02-448F-3R. These differentially expressed genes were activated during the early infection process, before the hypersensitive response or fungal growth inhibition occurred. Our analysis provides a list of candidate signaling components, which can be analyzed for function in Rpg1-mediated disease resistance.

Wheat cells accumulate a syringyl-rich lignin during the hypersensitive resistance response

The stem rust fungus Puccinia graminis f.sp. tritici is an obligately biotrophic pathogen attacking wheat ( Triticum aestivum). In compatible host/pathogen-interactions, the fungus participates in the host’s metabolism by establishing functional haustoria in the susceptible plant cells. In highly resistant wheat cultivars, fungal attack is stopped by a hypersensitive response of penetrated host cells. This mechanism of programmed cell death of single plant cells is accompanied by the intracellular accumulation of material with UV-fluorescence typical of phenolic compounds. A similar reaction can be induced in healthy wheat leaves by the application of a rust-derived elicitor. We analysed the biochemical composition of this defense-induced phenolic material. Contents of total soluble and cell wall esterified and etherified phenolic acids were determined in rust-inoculated and elicitor-treated leaves of the fully susceptible wheat cultivar Prelude and its highly resistant, near-isogenic line Prelude-Sr5. While no resistance-related changes occured in any of these fractions, the lignin content as determined by the thioglycolic acid and the acetyl bromide methods increased after elicitor treatment. Nitrobenzene oxidation revealed that the entire increase can be explained by an increase in syringyl units only. These biochemical data were confirmed by fluorescence emission spectra analyses which indicated a defense-induced enrichment of syringyl lignin for cell wall samples both from elicitor-treated wheat leaves and single host cells undergoing a hypersensitive response upon fungal penetration.

Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley

Rpg1 is a stem rust resistance gene that has protected barley from severe losses for over 60 years in the US and Canada. It confers resistance to many, but not all, pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. A fast neutron induced deletion mutant, showing susceptibility to stem rust pathotype Pgt-MCC, was identified in barley cv. Morex, which carries Rpg1. Genetic and Rpg1 mRNA and protein expression level analyses showed that the mutation was a suppressor of Rpg1 and was designated Rpr1 (Required for P. graminis resistance). Genome-wide expression profiling, using the Affymetrix Barley1 GeneChip containing approximately 22,840 probe sets, was conducted with Morex and the rpr1 mutant. Of the genes represented on the Barley1 microarray, 20 were up-regulated and 33 were down-regulated by greater than twofold in the mutant, while the Rpg1 mRNA level remained constant. Among the highly down-regulated genes (greater than fourfold), genomic PCR, RT-PCR and Southern analyses identified that three genes (Contig4901_s_at, HU03D17U_s_at, and Contig7061_s_at), were deleted in the rpr1 mutant. These three genes mapped to chromosome 4(4H) bin 5 and co-segregated with the rpr1-mediated susceptible phenotype. The loss of resistance was presumed to be due to a mutation in one or more of these genes. However, the possibility exists that there are other genes within the deletions, which are not represented on the Barley1 GeneChip. The Rpr1 gene was not required for Rpg5- and rpg4-mediated stem rust resistance, indicating that it shows specificity to the Rpg1-mediated resistance pathway.

Subcellular localization and functions of the barley stem rust resistance receptor-like serine/threonine-specific protein kinase Rpg1.

The Rpg1 gene confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici and has protected barley from serious disease losses for over 60 years. Rpg1 encodes a constitutively expressed protein with two tandem kinase domains. Fractionation by differential centrifugation and aqueous two-phase separation of the microsome proteins located Rpg1 mainly in the cytosol but also in the plasma membrane and intracellular membranes. Recombinant Rpg1 autophosphorylates in vitro intramolecularly only serine and threonine amino acids with a preference for Mn(2+) cations and a K(m) of 0.15 and a V(max) of 0.47 nmol.min(-1).mg(-1) protein. The inability of wild-type Rpg1 to transphosphorylate a recombinant Rpg1 inactivated by site-directed mutation confirmed that Rpg1 autophosphorylation proceeds exclusively via an intramolecular mechanism. Site-directed mutagenesis of the two adjacent lysine residues in the ATP anchor of the two-kinase domains established that the first of the two tandem kinase domains is nonfunctional and that lysine 461 of the second domain is the catalytically active residue. Transgenic barley, expressing Rpg1 mutated in either the kinase 1 or 2 domains, were fully susceptible to P. graminis f. sp. tritici revealing requirement of both kinase domains for resistance. In planta-expressed Rpg1 mutant protein confirmed that mutation in domain 2, but not 1, rendered the protein incapable of autophosphorylation.

Structure and expression of the barley stem rust resistance gene Rpg1 messenger RNA

The barley stem rust resistance gene Rpg1 confers resistance to many pathotypes of the stem rust fungus Puccinia graminis f. sp. tritici. Rpg1 was recently cloned by a map-based approach from the barley cultivar Morex. Haplotype sequencing and transformation of a susceptible barley cultivar were employed to prove its identity. As a first step in understanding the molecular mechanism of action of Rpg1, we characterized the structure and transcription of the Rpg1 messenger RNA. Several alternative splice forms were identified, some of which could be translated into a predicted full-length protein product. The expression of the gene occurred in all plant organs and developmental stages analyzed. However, leaf epidermis exhibited ca. 30-fold higher Rpg1 expression than the whole leaf. Expression of Rpg1 was not induced by infection of barley cultivar Morex seedlings with the incompatible P. graminis f. sp. tritici pathotype MCC, while some defense-related genes, such as barley β-1,3-glucanases and PR1a, were strongly induced.

A novel gene for rust resistance

The Rpg1 gene, which has provided North American cultivars of barley with resistance to the stem rust fungus Puccinia graminis f.sp. tritici for more than 60 years, has been cloned. A single copy of the gene can confer resistance to a susceptible barley variety. Although unexplained, the progeny are consistently more resistant than the variety from which the gene was obtained. The gene might represent a new class of plant resistance genes.

Genetically engineered stem rust resistance in barley using the Rpg1 gene.

The stem-rust-susceptible barley cv. Golden Promise was transformed by Agrobacterium-mediated transformation of immature zygotic embryos with the Rpg1 genomic clone of cv. Morex containing a 520-bp 5' promoter region, 4,919-bp gene region, and 547-bp 3' nontranscribed sequence. Representatives of 42 transgenic barley lines obtained were characterized for their seedling infection response to pathotype Pgt-MCC of the stem rust fungus Puccinia graminis f. sp. tritici. Golden Promise was converted from a highly susceptible cultivar into a highly resistant one by transformation with the dominant Rpg1 gene. A single copy of the gene was sufficient to confer resistance against stem rust, and progenies from several transformants segregated in a 3:1 ratio for resistancesusceptibility as expected for Mendelian inheritance. These results unequivocally demonstrate that the DNA segment isolated by map-based cloning is the functional Rpg1 gene for stem rust, resistance. One of the remarkable aspects about the transformants is that they exhibit a higher level of resistance than the original sources of Rpg1 (cvs. Chevron and Peatland). In most cases, the Golden Promise transformants exhibited a highly resistant reaction where no visible sign of infection was evident. Hypersensitive necrotic "fleck" reactions were also observed, but less frequently. With both infection types, pathogen sporulation was prevented. Southern blot and RT-PCR analysis revealed that neither Rpg1 gene copy number nor expression levels could account for the increased resistance observed in Golden Promise transformants. Nevertheless, this research demonstrates that stem-rust-susceptible barley can be made resistant by transformation with the cloned Rpg1 gene.

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.

Anthranilate Synthase and Chorismate Mutase Activities in Stem Rust-Inoculated and Elicitor-Treated Resistant, Moderately Resistant, and Susceptible Near-Isogenic Wheat Lines

Abstract Anthranilate synthase and chorismate mutase activities which control the flow of substrate from chorismate into the tryptophan pathway and into the phenylalanine/tyrosine pathway, respectively, were examined in three near isogenic wheat lines of Triticum aestivum L. (cv. Prelude Sr 5, highly resistant to stem rust infection; cv. Prelude Sr 24, moderately resistant; cv. Prelude srx, susceptible). The activities of both enzymes were found to increase in re­sponse to inoculation with the stem rust fungus Puccinia graminis f. sp. tritici or treatment with Pgt elicitor. Thus, both the tryptophan branch and the phenylalanine branch appear to contribute to the resistance response in wheat leaves. Only the cytosolic but not the plastidic fraction of the enzyme activities appears to be affected by fungal infection or elicitor treat­ment. Some differences with respect to degree and time dependency of enzyme activation were noticed between the three wheat lines following inoculation but not after treatment with the Pgt elicitor.

A β-1,3-D-endo-mannanase from culture filtrates of the hyperparasites Verticillium lecanii and Aphanocladium album that specifically lyses the germ pore plug from uredospores of Puccinia graminis f.sp. tritici

The hyperparasites Verticillium lecanii (Zimm.) Viegas syn. Verticillium hemileiae and Aphanocladium album (PreuB) W. Gams are capable of penetrating the hyphal walls and uredospores of the stem rust fungus Puccinia graminis f.sp. tritici, their potential host. The culture filtrates of both hyperparasites contain an array of polysaccharide-degrading enzymes that in concert, digest the walls of uredospores completely. We have now isolated an enzyme with β-1,3-D-endo-mannanase activity that is secreted by both hyperparasites into the culture medium in the presence of autoclaved walls of uredospores as a carbon source. The β-mannanase predominately lyses the germ pore plugs of uredospores as shown by electron scanning microscopy. The enzyme has an apparent molecular mass of 19 000 Da as revealed by medium pressure liquid chromatography using a Superose 12 gel filtration column. An enzyme with identical specificity was found in dormant and soaked uredospores. Since opening of the germ pore plug is a very early and crucial step in germination of uredospores, the implication of β-mannanases in the germination process is discussed. Key words: Aphanocladium album, germination, β-mannanase, hyperparasitism, Puccinia graminis, Verticillium lecanii.