Qingmei Han

High genome heterozygosity and endemic genetic recombination in the wheat stripe rust fungus

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat. Here we report a 110-Mb draft sequence of Pst isolate CY32, obtained using a ‘fosmid-to-fosmid’ strategy, to better understand its race evolution and pathogenesis. The Pst genome is highly heterozygous and contains 25,288 protein-coding genes. Compared with non-obligate fungal pathogens, Pst has a more diverse gene composition and more genes encoding secreted proteins. Re-sequencing analysis indicates significant genetic variation among six isolates collected from different continents. Approximately 35% of SNPs are in the coding sequence regions, and half of them are non-synonymous. High genetic diversity in Pst suggests that sexual reproduction has an important role in the origin of different regional races. Our results show the effectiveness of the ‘fosmid-to-fosmid’ strategy for sequencing dikaryotic genomes and the feasibility of genome analysis to understand race evolution in Pst and other obligate pathogens.

Cloning and characterization of a wheat β-1,3-glucanase gene induced by the stripe rust pathogen Puccinia striiformis f. sp. tritici

Abstractβ-1,3-Glucanases are a group of pathogenesis-related proteins that have been reported to be involved in plant defense against pathogens in many other plant-pathogen systems. However, it was not clear if these genes play similar role in wheat (Triticum aestivum L.) against Puccinia striiformis f. sp. tritici (Pst), the stripe rust pathogen. To investigate the role of β-1,3-glucanase (EC 3.2.1.39) in the resistance response of wheat (cv. Suwon 11) to stripe rust, a wheat β-1,3-glucanase gene induced by Pst, designated as TaGlu, was cloned and characterized. TaGlu was predicted to encode a basic protein of 334 amino acids. Quantitative real-time PCR analyses revealed that the transcription of TaGlu was induced during both compatible and incompatible interactions with Pst, but the transcription level was much higher in the incompatible interaction than that in the compatible interaction. TaGlu also showed noticeable induction of gene expression in young green leaf tissues treated with salicylic acid, methyl jasmonate or ethylene. Immunogold labeling assays showed that the enzyme were localized mainly in the host cell wall and over the extrahaustorial matrix, and the labeling densities were found significantly higher in the incompatible interaction than those in the compatible interaction.

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

BackgroundNon-host resistance (NHR) confers plant species immunity against the majority of microbial pathogens and represents the most robust and durable form of plant resistance in nature. As one 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 the mechanism of this kind of effective defense in legumes to these non-host pathogens.ResultsIn this study, the basis of NHR in broad bean (Vicia faba L.) against the wheat stripe rust pathogen, Puccinia striiformis f. sp. tritici (Pst), was characterized. No visible symptoms were observed on broad bean leaves inoculated with Pst. Microscopic observations showed that successful location of stomata and haustoria formation were significantly reduced in Pst infection of broad bean. Attempted infection induced the formation of papillae, cell wall thickening, production of reactive oxygen species, callose deposition and accumulation of phenolic compounds in plant cell walls. The few Pst haustoria that did form in broad bean cells 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 up-regulated during the Pst infection.ConclusionsThe results indicate that NHR in broad bean against Pst results from a continuum of layered defenses, 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 information for further determination of resistance mechanisms in broad bean to rust fungi, especially the adapted important broad bean rust pathogen, Uromyces viciae-fabae, because of strong similarity and association between NHR of plants to unadapted pathogens and basal resistance of plants to adapted pathogens.

Histological and cytological characterization of adult plant resistance to wheat stripe rust

AbstractWheat cultivar Xingzi 9104 (XZ) possesses adult plant resistance (APR) to stripe rust caused by Puccinia striiformis f. sp. tritici (Pst). In this study, histological and cytological experiments were conducted to elucidate the mechanisms of APR in XZ. The results of leaf inoculation experiments indicated that APR was initiated at the tillering stage, gradually increased as the plant aged and highly expressed after boot stage. The histology and oxidative burst in infected leaves of plants at seedling, tillering and boot stages were examined using light microscopic and histochemical methods. Subcellular changes in the host–pathogen interactions during the seedling and boot stages were analyzed by transmission electron microscopy. The results showed that haustorium formation was retarded in the adult plants and that the differentiation of secondary intercellular hyphae was significantly inhibited, which decreased the development of microcolonies in the adult plants, especially in plants of boot stage. The expression of APR to stipe rust during wheat development was clearly associated with extensive hypersensitive cell death of host cells and localized production of reactive oxygen species, which coincided with the restriction of fungal growth in infection sites in adult plants. At the same time, cell wall-related resistance in adult plants prevented ingression of haustorial mother cells into plant cells. Haustorium encasement was coincident with malformation or death of haustoria. The results provide useful information for further determination of mechanisms of wheat APR to stripe rust. Key message The expression of APR to stipe rust in wheat cultivar Xingzi 9104 (XZ) was clearly associated with extensive hypersensitive cell death of host cells and the localized production of reactive oxygen species.

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 H2O2 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.

Cytological and molecular characterization of non-host resistance in Arabidopsis thaliana against wheat stripe rust

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. We report the use of the non-host plant Arabidopsis thaliana to identify the basis of resistance to Pst at the cytological and molecular levels. No visible symptoms were observed on Arabidopsis leaves inoculated with Pst. Microscopic observations showed that significantly reduced numbers of Pst urediospores had successfully achieved penetration in Arabidopsis compared with those in wheat. There were significant differences in the frequency of stomatal penetration but not in fungal growth among different Pst races in Arabidopsis. The fungus failed to successfully form haustoria in Arabidopsis and attempted infection induced an active response including accumulation of phenolic compounds and callose deposition in plant cells. A set of defence-related genes were also up regulated during the Pst infection. Compared with wild type plants, increased fungal growth was observed in an npr1-1 mutant and in NahG transformed plants, which both are insensitive to salicylic acid. However, treatment of Arabidopsis plants with cytochalasin B, an inhibitor of actin microfilament polymerization, did not increase susceptibility to Pst. Our results demonstrate that Arabidopsis can be used to study mechanisms of non-host resistance to wheat stripe rust, and highlight the significance of participation of salicylic acid in non-host resistance to rust fungi.

Biological control of oilseed rape Sclerotinia stem rot by Bacillus subtilis strain Em7

In the present study, the endophytic bacterium Bacillus subtilis strain Em7 (GU258545.1) was evaluated as a biological control agent for Sclerotinia sclerotiorum on oilseed rape. In petri dish, strain Em7 not only strongly inhibited pathogen mycelium growth but also germination of sclerotia at concentrations between 109 and 1011 colony forming unit (CFU)·ml−1. Scanning electron microscopy and transmission electron microscopy studies revealed that in the presence of strain Em7, hyphae of S. sclerotiorum showed leakage and disintegration of hyphal cytoplasm. Furthermore, the strain Em7 showed a broad antifungal spectrum on mycelium growth of numerous important plant pathogenic fungi. Light microscopic observations revealed that strain Em7 caused morphological alterations including increased branching, swelling and collapse of cytoplasm. In the greenhouse, spray treatments of cell suspensions of strain Em7 (1×109 CFU·ml−1) reduced leaf and stem rot incidence and severity in the seedling and blossom stage. The control efficacy was higher when strain Em7 cell suspension was applied one day prior to inoculation of the pathogen than after inoculation. Three-year field trials showed that two applications of strain Em7 cell suspension at blossom stage significantly reduced disease incidence and severity by 50–70%. There was no significant difference in control efficacy among treatments with strain Em7 cell suspension and the fungicides containing carbendazim or tebuconazole (P = 0.05). Thus, our results strongly suggest that B. subtilis strain Em7 is a promising biological control agent for control of oilseed rape Sclerotinia stem rot.

Studies on the Infection, Colonization, and Movement of Pseudomonas syringae pv. actinidiae in Kiwifruit Tissues Using a GFPuv-Labeled Strain

Kiwifruit bacterial canker, an economically important disease caused by Pseudomonas syringae pv. actinidiae (Psa), has caused severe losses in all major areas of kiwifruit cultivation. Using a GFPuv-labeled strain of Psa, we monitored the invasion, colonization, and movement of the pathogen in kiwifruit twigs, leaves and veins. The pathogen can invade twigs through both wounds and natural openings; the highest number of Psa is obtained in cut tissues. We determined that, following spray inoculation, Psa-GFPuv could infect leaves and cause lesions in the presence and absence of wounds. Light and transmission electron microscopic observations showed that bacterial cells colonize both phloem and xylem vessels. Bacterial infection resulted in marked alterations of host tissues including the disintegration of organelles and degeneration of protoplasts and cell walls. Furthermore, low temperature was conducive to colonization and movement of Psa-GFPuv in kiwifruit tissues. Indeed, the pathogen migrated faster at 4°C than at 16°C or 25°C in twigs. However, the optimum temperature for colonization and movement of Psa in leaf veins was 16°C. Our results, revealing a better understanding of the Psa infection process, might contribute to develop more efficacious disease management strategies.

PsANT, the adenine nucleotide translocase of Puccinia striiformis, promotes cell death and fungal growth

Adenine nucleotide translocase (ANT) is a constitutive mitochondrial component that is involved in ADP/ATP exchange and mitochondrion-mediated apoptosis in yeast and mammals. However, little is known about the function of ANT in pathogenic fungi. In this study, we identified an ANT gene of Puccinia striiformis f. sp. tritici (Pst), designated PsANT. The PsANT protein contains three typical conserved mitochondrion-carrier-protein (mito-carr) domains and shares more than 70% identity with its orthologs from other fungi, suggesting that ANT is conserved in fungi. Immuno-cytochemical localization confirmed the mitochondrial localization of PsANT in normal Pst hyphal cells or collapsed cells. Over-expression of PsANT indicated that PsANT promotes cell death in tobacco, wheat and fission yeast cells. Further study showed that the three mito-carr domains are all needed to induce cell death. qRT-PCR analyses revealed an in-planta induced expression of PsANT during infection. Knockdown of PsANT using a host-induced gene silencing system (HIGS) attenuated the growth and development of virulent Pst at the early infection stage but not enough to alter its pathogenicity. These results provide new insight into the function of PsANT in fungal cell death and growth and might be useful in the search for and design of novel disease control strategies.

Endophytic Bacillus subtilis Strain E1R-J Is a Promising Biocontrol Agent for Wheat Powdery Mildew

In this study, the biocontrol efficacies of 14 endophytic bacterial strains were tested against Blumeria graminis f. sp. tritici (Bgt) in pot experiments under greenhouse conditions. Bacillus subtilis strain E1R-j significantly reduced disease index and exhibited the best control (90.97%). When different formulations of E1R-j were sprayed 24 h before Bgt inoculation, fermentation liquid without bacterial cell and crude protein suspension displayed the similar effects; and they reduced disease index more than bacterial cell suspension (109 cfu mL−1) and fermentation liquid without protein. The control effects were not significantly different between 1011 and 109 cfu mL−1 of bacterial cell suspension but were higher than 107 cfu mL−1. Further observations showed that conidial germination and appressorial formation of Bgt were retarded by spraying E1R-j 24 h before Bgt inoculation. Compared with the water check, conidial germination and appressorial formation were decreased by 43.3% and 42.7%, respectively. In the treatment with E1R-j, the number of houstoria significantly reduced and the speed of mycelial extension was slowed down in the wheat leaves. Scanning electron microscopy observation revealed that E1R-j significantly suppressed the conidial germination and caused rupture and deformation of germ tubes. On the surface of wheat leaves, mycelia and conidiophores became shrinking.