Cytological and molecular analysis of the Hordeum vulgare-Puccinia triticina nonhost interaction

Cultivated barley, Hordeum vulgare L., is considered to be a nonhost or intermediate host species for the wheat leaf rust fungus Puccinia triticina. Here, we have investigated, at the microscopic and molecular levels, the reaction of barley cultivars to wheat leaf rust infection. In the nonhost resistant cultivar Cebada Capa, abortion of fungal growth occurred at both pre- and posthaustorial stages, suggesting that defense genes are expressed throughout the development of the inappropriate fungus during the nonhost resistance reaction. In the two barley lines L94 and Bowman, a low level of prehaustorial resistance to P. triticina was observed and susceptibility was comparable to that of wheat control plants. Suppression subtractive hybridization was used to identify genes that are differentially expressed during the nonhost resistance reaction in Cebada Capa as well as during the successful establishment of the inappropriate wheat leaf rust fungus in L94. Northern analysis indicated that two candidate genes, including a barley ortholog of the rice resistance gene Xa21, are putatively involved in nonhost and non-race-specific resistance reactions. In addition, a new gene that is specifically induced during the successful development of the inappropriate fungus P. triticina in barley has been identified.     

Characterization of nonhost resistance of Arabidopsis to the Asian soybean rust

Asian soybean rust (ASR), caused by Phakopsora pachyrhizi, is a devastating disease of soybean. We report the use of the nonhost plant Arabidopsis thaliana to identify the genetic basis of resistance to P. pachyrhizi. Upon attack by P. pachyrhizi, epidermal cells of wild-type Arabidopsis accumulated H2O2, which likely orchestrates the frequently observed epidermal cell death. However, even when epidermal cell death occurred, fungal hyphae grew on and infection was terminated at the mesophyll boundary. These events were associated with expression of PDF1.2, suggesting that P. pachyrhizi, an ostensible biotroph, mimics aspects of a necrotroph. Extensive colonization of the mesophyll occurred in Arabidopsis pen mutants with defective penetration resistance. Although haustoria were found occasionally in mesophyll cells, the successful establishment of biotrophy failed, as evidenced by the cessation of fungal growth. Double mutants affected in either jasmonic acid or salicylic acid signaling in the pen3-1 background revealed the involvement of both pathways in nonhost resistance (NHR) of Arabidopsis to P. pachyrhizi. Interestingly, expression of AtNHL10, a gene that is expressed in tissue undergoing the hypersensitive response, was only triggered in infected pen3-1 mutants. Thus, a suppression of P. pachyrhizi-derived effectors by PEN3 can be inferred. Our results demonstrate that Arabidopsis can be used to study mechanisms of NHR to ASR.     

Interspecies gene transfer provides soybean resistance to a fungal pathogen

Fungal pathogens pose a major challenge to global crop production. Crop varieties that resist disease present the best defence and offer an alternative to chemical fungicides. Exploiting durable nonhost resistance (NHR) for crop protection often requires identification and transfer of NHR‐linked genes to the target crop. Here, we identify genes associated with NHR of Arabidopsis thaliana to Phakopsora pachyrhizi, the causative agent of the devastating fungal disease called Asian soybean rust. We transfer selected Arabidopsis NHR‐linked genes to the soybean host and discover enhanced resistance to rust disease in some transgenic soybean lines in the greenhouse. Interspecies NHR gene transfer thus presents a promising strategy for genetically engineered control of crop diseases.     

Tracking wheat rust on a continental scale

The rusts of wheat are important fungal plant pathogens that can be disseminated thousands of kilometers across continents and oceans by wind. Rusts are obligate parasites that interact with resistance genes in wheat in a gene-for-gene manner. New races of rust develop by mutation and selection for virulence against rust resistance genes in wheat. In recent years, new races of wheat leaf rust, wheat stripe rust, and wheat stem rust have been introduced into wheat production areas in different continents. These introductions have complicated efforts to develop wheat cultivars with durable rust resistance and have reduced the number of effective rust-resistance genes that are available for use. The migration patterns of wheat rusts are characterized by identifying their virulence against important rust resistance genes in wheat and by the use of molecular markers.     

Histological and molecular studies of the non-host interaction between wheat and Uromyces fabae

Non-host resistance (NHR) confers plant species immunity against the majority of microbes. As an important crop, wheat can be damaged by several Puccinia species but is immune to all Uromyces species. Here, we studied the basis of NHR in wheat against the broad bean rust pathogen Uromyces fabae (Uf). In the wheat-Uf interaction, microscopic observations showed that urediospores germinated efficiently on wheat leaves. However, over 98% of the germ tubes failed to form appressoria over stomata. For the few that invaded through stomata, the majority of them failed to penetrate wheat mesophyll cells. At 96 hours after inoculation, less than 4% of the Uf infection units that had entered the mesophyll tissue formed haustoria. Attempted penetration by haustorium mother cells induced the thickening of cell wall and the formation of papillae in plant cells, which arrested the development or growth of Uf penetration pegs. For the Uf haustoria formed in wheat cells, they were encased in callose-like materials and did not elicit hypersensitive response. Localized accumulation of H(2)O(2) were observed in plant cell walls, papillae and encasement of haustoria during the wheat-Uf interaction. Furthermore, quantitative RT-PCR analysis showed that several genes involved in basal resistance and oxidative stress responses were up-regulated during Uf infection. In conclusion, our study revealed the cytological and molecular bases of NHR in wheat against the non-adapted rust fungus Uf, and highlighted the significance of papilla production in the prehaustorial NHR.     

Role of topography sensing for infection-structure differentiation in cereal rust fungi

Over 90% of the germ tubes of Puccinia graminis tritici (wheat stem rust) and Puccinia hordei (barley brown rust) differentiate appressoria on encountering stomata.There has been controversy as to the role of host topographical signals in the highly precise and efficient induction of these infection structures over stomata by cereal rusts. In the present study, polystyrene replicas of microfabricated silicon wafers, bearing precise microtopographies of defined dimensions, were used to investigate the influence of ridge spacing and height on infection-structure induction by P. graminis tritici and P. hordei. It was found that artificial topographical signals alone can induce a reproducibly high percentage (83–86%) of germ tubes to differentiate infection structures. Multiple, closely spaced (1.5 μm) ridges which were 2.0 μm high provided the most inductive topography. Differentiation on flat surfaces and over single ridges was < 4%. Appressorium induction commonly initiated a cascade of differentiation events involving the formation of infection pegs, vesicles, infection hyphae, and occasionally haustorial mother cells. It is suggested that the close spacing of cell junctions associated with the dumbbell-shaped guard cells of cereal stomatal complexes provide inductive signals for infection-structure formation by cereal rusts in vivo. Received: 17 October 1996 / Accepted: 5 December 1996     

High diversity of genes for nonhost resistance of barley to heterologous rust fungi

Inheritance studies on the nonhost resistance of plants would normally require interspecific crosses that suffer from sterility and abnormal segregation. Therefore, we developed the barley-Puccinia rust model system to study, using forward genetics, the specificity, number, and diversity of genes involved in nonhost resistance. We developed two mapping populations by crossing the line SusPtrit, with exceptional susceptibility to heterologous rust species, with the immune barley cultivars Vada and Cebada Capa. These two mapping populations along with the Oregon Wolfe Barley population, which showed unexpected segregation for resistance to heterologous rusts, were phenotyped with four heterologous rust fungal species. Positions of QTL conferring nonhost resistance in the three mapping populations were compared using an integrated consensus map. The results confirmed that nonhost resistance in barley to heterologous rust species is controlled by QTL with different and overlapping specificities and by an occasional contribution of an R-gene for hypersensitivity. In each population, different sets of loci were implicated in resistance. Few genes were common between the populations, suggesting a high diversity of genes conferring nonhost resistance to heterologous pathogens. These loci were significantly associated with QTL for partial resistance to the pathogen Puccinia hordei and with defense-related genes.     

Characterization of protein kinase PsSRPKL,a novel pathogenicity factor in the wheat stripe rust fungus

As in other eukaryotes, protein kinases (PKs) are generally evolutionarily conserved and play major regulatory roles in plant pathogenic fungi. Many PKs have been proven to be important for pathogenesis in model fungal plant pathogens, but little is currently known about their roles in the pathogenesis of cereal rust fungi, devastating pathogens in agriculture worldwide. Here, we report on an in planta highly induced PK gene PsSRPKL from the wheat stripe rust fungus Puccinia striiformis f. sp. tritici (Pst), one of the most important cereal rust fungi. PsSRPKL belongs to a group of PKs that are evolutionarily specific to cereal rust fungi. It shows a high level of intraspecies polymorphism in the kinase domains and directed green fluorescent protein chimers to plant nuclei. Overexpression of PsSRPKL in fission yeast induces aberrant cell morphology and a decreased resistance to environmental stresses. Most importantly, PsSRPKL is proven to be an important pathogenicity factor responsible for fungal growth and responses to environmental stresses, therefore contributing significantly to Pst virulence in wheat. We hypothesize that cereal rust fungi have developed specific PKs as pathogenicity factors for adaptation to their host species during evolution. Thus, our findings provide significant insights into pathogenicity and virulence evolution in cereal rust fungi.     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

Characterization of genetic loci conferring adult plant resistance to leaf rust and stripe rust in spring wheat.

Leaf (brown) and stripe (yellow) rusts, caused by Puccinia triticina and Puccinia striiformis, respectively, are fungal diseases of wheat (Triticum aestivum) that cause significant yield losses annually in many wheat-growing regions of the world. The objectives of our study were to characterize genetic loci associated with resistance to leaf and stripe rusts using molecular markers in a population derived from a cross between the rust-susceptible cultivar 'Avocet S' and the resistant cultivar 'Pavon76'. Using bulked segregant analysis and partial linkage mapping with AFLPs, SSRs and RFLPs, we identified 6 independent loci that contributed to slow rusting or adult plant resistance (APR) to the 2 rust diseases. Using marker information available from existing linkage maps, we have identified additional markers associated with resistance to these 2 diseases and established several linkage groups in the 'Avocet S' x 'Pavon76' population. The putative loci identified on chromosomes 1BL, 4BL, and 6AL influenced resistance to both stripe and leaf rust. The loci on chromosomes 3BS and 6BL had significant effects only on stripe rust, whereas another locus, characterized by AFLP markers, had minor effects on leaf rust only. Data derived from Interval mapping indicated that the loci identified explained 53% of the total phenotypic variation (R2) for stripe rust and 57% for leaf rust averaged across 3 sets of field data. A single chromosome recombinant line population segregating for chromosome 1B was used to map Lr46/Yr29 as a single Mendelian locus. Characterization of slow-rusting genes for leaf and stripe rust in improved wheat germplasm would enable wheat breeders to combine these additional loci with known slow-rusting loci to generate wheat cultivars with higher levels of slow-rusting resistance.     

The durable wheat disease resistance gene Lr34 confers common rust and northern corn leaf blight resistance in maize

Maize (corn) is one of the most widely grown cereal crops globally. Fungal diseases of maize cause significant economic damage by reducing maize yields and by increasing input costs for disease management. The most sustainable control of maize diseases is through the release and planting of maize cultivars with durable disease resistance. The wheat gene Lr34 provides durable and partial field resistance against multiple fungal diseases of wheat, including three wheat rust pathogens and wheat powdery mildew. Because of its unique qualities, Lr34 became a cornerstone in many wheat disease resistance programmes. The Lr34 resistance is encoded by a rare variant of an ATP‐binding cassette (ABC) transporter that evolved after wheat domestication. An Lr34‐like disease resistance phenotype has not been reported in other cereal species, including maize. Here, we transformed the Lr34 resistance gene into the maize hybrid Hi‐II. Lr34‐expressing maize plants showed increased resistance against the biotrophic fungal disease common rust and the hemi‐biotrophic disease northern corn leaf blight. Furthermore, the Lr34‐expressing maize plants developed a late leaf tip necrosis phenotype, without negative impact on plant growth. With this and previous reports, it could be shown that Lr34 is effective against various biotrophic and hemi‐biotrophic diseases that collectively parasitize all major cereal crop species.     

Accumulation of genes for susceptibility to rust fungi for which barley is nearly a nonhost results in two barley lines with extreme multiple susceptibility

Nonhost resistance is the most common type of resistance in plants. Understanding the factors that make plants susceptible or resistant may help to achieve durably effective resistance in crop plants. Screening of 109 barley (Hordeum vulgare L.) accessions in the seedling stage indicated that barley is a complete nonhost to most of the heterologous rust fungi studied, while it showed an intermediate status with respect to Puccinia triticina, P. hordei-murini, P. hordei-secalini, P. graminis f. sp. lolii and P. coronata ff. spp. avenae and holci. Accessions that were susceptible to a heterologous rust in the seedling stage were much more or completely resistant at adult plant stage. Differential interaction between barley accessions and heterologous rust fungi was found, suggesting the existence of rust-species-specific resistance. In particular, many landrace accessions from Ethiopia and Asia, and naked-seeded accessions, tended to be susceptible to several heterologous rusts, suggesting that some resistance genes in barley are effective against more than one heterologous rust fungal species. Some barley accessions had race-specific resistance against P. hordei-murini. We accumulated genes for susceptibility to P. triticina and P. hordei-murini in two genotypes called SusPtrit and SusPmur, respectively. In the seedling stage, these accessions were as susceptible as the host species to the target rusts. They also showed unusual susceptibility to other heterologous rusts. These two lines are a valuable asset to further experimental work on the genetics of resistance to heterologous rust fungi.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00425-004-1319-1Abbreviations ff. spp Formae speciales - RIL Recombinant inbred line - DC Double cross - DC-S Progeny produced by selfing of double-cross plants     

Characterization of non-host resistance in broad bean to the wheat stripe rust pathogen

ABSTRACT: BACKGROUND: Non-host resistance (NHR) confers plant species immunity against the majority of microbialpathogens and represents the most robust and durable form of plant resistance in nature. Asone of the main genera of rust fungi with economic and biological importance, Puccinia infects almost all cereals but is unable to cause diseases on legumes. Little is known about themechanism of this kind of effective defense in legumes to these non-host pathogens. RESULTS: In this study, the basis of NHR in broad bean (Vicia faba L.) against the wheat stripe rustpathogen, Puccinia striiformis f. sp. tritici (Pst), was characterized. No visible symptomswere observed on broad bean leaves inoculated with Pst. Microscopic observations showedthat successful location of stomata and haustoria formation were significantly reduced in Pstinfection of broad bean. Attempted infection induced the formation of papillae, cell wallthickening, production of reactive oxygen species, callose deposition and accumulation ofphenolic compounds in plant cell walls. The few Pst haustoria that did form in broad beancells were encased in reactive oxygen and callose materials and those cells elicited cell death.Furthermore, a total of seven defense-related genes were identified and found to be upregulatedduring the Pst infection. CONCLUSIONS: The results indicate that NHR in broad bean against Pst results from a continuum of layereddefenses, including basic incompatibility, structural and chemical strengthening of cell wall,posthaustorial hypersensitive response and induction of several defense-related genes,demonstrating the multi-layered feature of NHR. This work also provides useful informationfor further determination of resistance mechanisms in broad bean to rust fungi, especially theadapted important broad bean rust pathogen, Uromyces viciae-fabae, because of strongsimilarities and association between NHR of plants to unadapted pathogens and basalresistance of plants to adapted pathogens.     

A mutagenesis-derived broad-spectrum disease resistance locus in wheat

Wheat leaf rust, stem rust, stripe rust, and powdery mildew caused by the fungal pathogens Puccinia triticina, P. graminis f. sp. tritici, P. striiformis f. sp. tritici, and Blumeria graminis f. sp. tritici, respectively, are destructive diseases of wheat worldwide. Breeding durable disease resistance cultivars rely largely on continually introgressing new resistance genes, especially the genes with different defense mechanisms, into adapted varieties. Here, we describe a new resistance gene obtained by mutagenesis. The mutant, MNR220 (mutagenesis-derived new resistance), enhances resistance to three rusts and powdery mildew, with the characteristics of delayed disease development at the seedling stage and completed resistance at the adult plant stage. Genetic analysis demonstrated that the resistance in MNR220 is conferred by a single semidominant gene mapped on the short arm of chromosome 2B. Gene expression profiling of several pathogenesis-related genes indicated that MNR220 has an elevated and rapid pathogen-induced response. In addition to its potential use in breeding for resistance to multiple diseases, high-resolution mapping and cloning of the disease resistance locus in MNR220 may lead to a better understanding of the regulation of defense responses in wheat.     

QTL mapping of adult-plant resistances to stripe rust and leaf rust in Chinese wheat cultivar Bainong 64

Stripe rust and leaf rust, caused by Puccinia striiformis Westend. f. sp. tritici Erikss. and P. triticina, respectively, are devastating fungal diseases of common wheat (Triticum aestivum L.). Chinese wheat cultivar Bainong 64 has maintained acceptable adult-plant resistance (APR) to stripe rust, leaf rust and powdery mildew for more than 10?years. The aim of this study was to identify quantitative trait loci/locus (QTL) for resistance to the two rusts in a population of 179 doubled haploid (DH) lines derived from Bainong 64?×?Jingshuang 16. The DH lines were planted in randomized complete blocks with three replicates at four locations. Stripe rust tests were conducted using a mixture of currently prevalent P. striiformis races, and leaf rust tests were performed with P. triticina race THTT. Leaf rust severities were scored two or three times, whereas maximum disease severities (MDS) were recorded for stripe rust. Using bulked segregant analysis (BSA) and simple sequence repeat (SSR) markers, five independent loci for APR to two rusts were detected. The QTL on chromosomes 1BL and 6BS contributed by Bainong 64 conferred resistance to both diseases. The loci identified on chromosomes 7AS and 4DL had minor effects on stripe rust response, whereas another locus, close to the centromere on chromosome 6BS, had a significant effect only on leaf rust response. The loci located on chromosomes 1BL and 4DL also had significant effects on powdery mildew response. These were located at the same positions as the Yr29/Lr46 and Yr46/Lr67 genes, respectively. The multiple disease resistance locus for APR on chromosome 6BS appears to be new. All three genes and their closely linked molecular markers could be used in breeding wheat cultivars with durable resistance to multiple diseases.     

条形柄锈菌(小麦条锈病菌)侵染结构体外形成的观察

通过荧光显微镜和扫描电镜分别对条形柄锈菌夏孢子在寄主植物-小麦叶表和非寄主植物-水稻叶表以及小麦穗部和茎秆上的萌发过程进行了观察。结果发现,夏孢子在小麦叶片体表萌发产生芽管后,可依次分化形成气孔下囊、初生菌丝与吸器母细胞;在小麦颖片、稃片及茎秆部位表面,同样可观察到病菌在体外分化形成吸器母细胞;并且在水稻叶片上也观察到病菌侵染结构存在体外分化现象。经荧光染色发现,条形柄锈菌在体外与在小麦组织中形成的侵染结构没有明显的差别。观察结果可为条形柄锈菌侵染结构的离体诱导与调控机理研究提供依据。     

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.     

Cryptic diversity and significant cophylogenetic signal detected by DNA barcoding the rust fungi (Pucciniaceae) of Cyperaceae–Juncaceae

Plants play important roles as habitat and food for a tremendous diversity of specialist animals and fungi. The disappearance of any plant species can lead to extinction cascades of its associated biota. In consequence, documenting the diversity and specificity of plant-associated organisms is of high practical relevance in biodiversity conservation. Here, we present the first large-scale molecular investigation into the diversity, host specificity, and cophylogenetic congruence of an especially rich plant–fungal association, the rust fungi (Pucciniaceae) of Cyperaceae and Juncaceae. Using the largest rust fungi DNA barcoding dataset published to date (252 sequences, 82 taxa), we reject the presence of a global ITS2-28S barcode gap, but find a local gap in Cyperaceae–Juncaceae rusts, and suggest the existence of many cryptic species in North America, with some broadly circumscribed species possibly corresponding to >10 cryptic species. We test previous hypotheses of correlations between the phylogenies of rust fungi and their Cyperaceae–Juncaceae hosts using a combination of global-fit and event-based cophylogenetic methods. A significant cophylogenetic signal is detected between rusts and their hosts, but the small number of cospeciations argues for preferential host jumps as the driving process behind these correlations. In addition, temporal congruence between the origin of major Carex clades and their rusts suggests that host diversification may have promoted parasite diversification. Finally, we discuss the relevance of rust infection patterns to the systematics of Cyperaceae, highlight some taxonomic problems uncovered by the analyses, and call attention to the promise of DNA barcoding for bridging knowledge gaps in poorly studied plant-associated microorganisms.