Yulin Cheng

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.

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.

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.

Wheat TaRab7 GTPase is part of the signaling pathway in responses to stripe rust and abiotic stimuli.

Small GTP-binding proteins function as regulators of specific intercellular fundamental biological processes. In this study, a small GTP-binding protein Rab7 gene, designated as TaRab7, was identified and characterized from a cDNA library of wheat leaves infected with Puccinia striiformis f. sp. tritici (Pst) the wheat stripe rust pathogen. The gene was predicted to encode a protein of 206 amino acids, with a molecular mass of 23.13 KDa and an isoeletric point (pI) of 5.13. Further analysis revealed the presence of a conserved signature that is characteristic of Rab7, and phylogenetic analysis demonstrated that TaRab7 has the highest similarity to a small GTP binding protein gene (BdRab7-like) from Brachypodium distachyon. Quantitative real-time PCR assays revealed that the expression of TaRab7 was higher in the early stage of the incompatible interactions between wheat and Pst than in the compatible interaction, and the transcription level of TaRab7 was also highly induced by environmental stress stimuli. Furthermore, knocking down TaRab7 expression by virus induced gene silencing enhanced the susceptibility of wheat cv. Suwon 11 to an avirulent race CYR23. These results imply that TaRab7 plays an important role in the early stage of wheat-stripe rust fungus interaction and in stress tolerance.

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.

TaEIL1, a wheat homologue of AtEIN3, acts as a negative regulator in the wheat-stripe rust fungus interaction

Transcription factors (TFs) play crucial roles in the transcriptional regulation of plant development and defence responses. Increasing evidence has implicated ETHYLENE INSENSITIVE3 (EIN3) in the plant defence response to pathogen infection and environmental stimuli. However, the role of EIN3 in wheat resistance to Puccinia striiformis f. sp. tritici (Pst) is not clear. Here, TaEIL1 was isolated by rapid amplification of cDNA ends (RACE) based on a sequence fragment from a wheat-Pst interaction cDNA library. The TaEIL1 protein contains a typical EIN3-binding domain, and transient expression analyses indicated that TaEIL1 is localized in the nucleus. Yeast one-hybrid assay revealed that TaEIL1 exhibits transcriptional activity, and its C-terminus is necessary for the activation of transcription. TaEIL1 transcripts were regulated by environmental stress stimuli and were decreased under salicylic acid (SA) treatment. When wheat leaves were challenged with Pst, the transcript level of TaEIL1 in the compatible interaction was approximately three times higher than that in the incompatible interaction. Knocking down TaEIL1 through the Barley stripe mosaic virus (BSMV) virus-induced gene silencing (VIGS) system attenuated the growth of Pst, with shortened hyphae and reduced hyphal branches, haustorial mother cells and colony size. Moreover, enhanced necrosis was triggered by the Pst avirulent race CYR23, indicating that the hypersensitive response was strengthened in TaEIL1-silenced wheat plants. Thus, the up-regulation of defence-related genes and increased sucrose abundance might contribute to the enhanced disease resistance of wheat to the virulent race CYR31. Taken together, the results suggested that the suppression of TaEIL1 transcripts could enhance the resistance of wheat to stripe rust fungus.

Two distinct Ras genes from Puccinia striiformis exhibit differential roles in rust pathogenicity and cell death.

Ras genes have been shown to regulate a variety of cellular processes in higher eukaryotes. However, much less is known about their function(s) in fungi, especially plant pathogenic fungi. Here, we report the identification and functional analysis of Ras genes from Puccinia striiformis f. sp. tritici (Pst), an important fungal pathogen in wheat production worldwide. Pst contains two Ras genes, PsRas1 and PsRas2, which share 48.6% similarity at the protein level and fall into two different phylogenetic clades. Both PsRas1 and PsRas2 have conserved protein sequences among different Pst isolates, but exhibit different transcript profiles during Pst infection. Silencing of PsRas1 or PsRas2 indicates that PsRas2 but not PsRas1 contributes significantly to rust pathogenicity. However, overexpression of PsRas1, but not PsRas2, promotes cell death in yeast and plants. Further studies show that all conserved domains of Ras GTPases in PsRas1 are needed to induce this cell death. In plants, PsRas1-triggered cell death shows similar characteristics as plant hypersensitive response. Our findings suggest that PsRas1 and PsRas2 take over different functions in rust pathogenicity and cell death, thus facilitating the understanding of cell death, pathogenic mechanisms of plant pathogenic fungi and the search for novel pathogen control strategies.

Cytological and molecular analysis of nonhost resistance in rice to wheat powdery mildew and leaf rust pathogens

Cereal powdery mildews caused by Blumeria graminis and cereal rusts caused by Puccinia spp. are constant disease threats that limit the production of almost all important cereal crops. Rice is an intensively grown agricultural cereal that is atypical because of its immunity to all powdery mildew and rust fungi. We analyzed the nonhost interactions between rice and the wheat powdery mildew fungus B. graminis f. sp. tritici (Bgt) and the wheat leaf rust fungus Puccinia triticina (Ptr) to identify the basis of nonhost resistance (NHR) in rice against cereal powdery mildew and rust fungi at cytological and molecular levels. No visible symptoms were observed on rice leaves inoculated with Bgt or Ptr. Microscopic observations showed that both pathogens exhibited aberrant differentiation and significantly reduced penetration frequencies on rice compared to wheat. The development of Bgt and Ptr was also completely arrested at early infection stages in cases of successful penetration into rice leaves. Attempted infection of rice by Bgt and Ptr induced similar defense responses, including callose deposition, accumulation of reactive oxygen species, and hypersensitive response in rice epidermal and mesophyll cells, respectively. Furthermore, a set of defense-related genes were upregulated in rice against Bgt and Ptr infection. Rice is an excellent monocot model for genetic and molecular studies. Therefore, our results demonstrate that rice is a useful model to study the mechanisms of NHR to cereal powdery mildew and rust fungi, which provides useful information for the development of novel and durable strategies to control these important pathogens.