Hongchang Zhang

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.

TaDAD2, a Negative Regulator of Programmed Cell Death, Is Important for the Interaction Between Wheat and the Stripe Rust Fungus

Defender against cell death (DAD) genes are known to function as negative regulators of cell death in animals. In plants, DAD orthologs are conserved but their role in cell death regulation is not well understood. Here, we report the characterization of the TaDAD2 gene in wheat. The predicted amino acid sequence of TaDAD2 contains typical structural features of DAD proteins, including a signal peptide, three transmembrane regions, and a subunit of oligosaccharyltransferase. Transcripts of TaDAD2 were detected in wheat leaves, culms, roots, florets, and spikelets. The expression level of TaDAD2 was reduced in the initial contact with the stripe rust fungus, subsequently induced and peaked at 18 h postinoculation (hpi), gradually reduced at 24 to 48 hpi, and restored to control level at 72 to 120 hpi. In addition, TaDAD2 exhibited positive transcriptional responses to abiotic stresses after the initial reduction at 1 hpi. Overexpression of TaDAD2 in tobacco leaves inhibited cell death. Furthermore, knocking down TaDAD2 expression by virus-induced gene silencing enhanced the susceptibility of wheat cv. Suwon11 to avirulent race CYR23 and reduced necrotic area at the infection sites. These results indicate that TaDAD2 may function as a suppressor of cell death in the early stages of wheat-stripe rust fungus interaction. However, it is dispensable for or plays an opposite role in hypersensitive response or cell death triggered by an avirulent race of stripe rust fungus at late-infection stages.

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.

Different non-host resistance responses of two rice subspecies, japonica and indica, to Puccinia striiformis f. sp. tritici

Key messageJaponicaandindicahave different non-host resistance (NHR) abilities toPuccinia striiformisf. sp.tritici(Pst), and hydrogen peroxide (H2O2) has a positive function in NHR tojaponicaagainstPst.AbstractNon-host interactions between Puccinia striiformis f. sp. tritici (Pst) and two rice subspecies were characterized using 23 rice varieties, including 11 japonica and 12 indica. Results showed that the infected fungal structures were easily produced in the leaves of indica, whereas only several substomatal vesicles and primary infection hyphae were observed in the leaves of japonica. This result indicated that indica is less resistant or more susceptible to Pst than japonica. Hydrogen peroxide accumulated in the initial phase of japonica–Pst interaction but not in indica–Pst interaction. A set of reactive oxygen species (ROS)-related genes was also induced in response to Pst infection, suggesting that ROS activation is one of the major mechanisms of non-host resistance of rice to Pst.

FgCDC14 regulates cytokinesis, morphogenesis, and pathogenesis in Fusarium graminearum

Members of Cdc14 phosphatases are common in animals and fungi, but absent in plants. Although its orthologs are conserved in plant pathogenic fungi, their functions during infection are not clear. In this study, we showed that the CDC14 ortholog is important for pathogenesis and morphogenesis in Fusarium graminearum. FgCDC14 is required for normal cell division and septum formation and FgCdc14 possesses phosphatase activity with specificity for a subset of Cdk‐type phosphorylation sites. The Fgcdc14 mutant was reduced in growth, conidiation, and ascospore formation. It was defective in ascosporogenesis and pathogenesis. Septation in Fgcdc14 was reduced and hyphal compartments contained multiple nuclei, indicating defects in the coordination between nuclear division and cytokinesis. Interestingly, foot cells of mutant conidia often differentiated into conidiogenous cells, resulting in the production of inter‐connected conidia. In the interphase, FgCdc14‐GFP localized to the nucleus and spindle‐pole‐body. Taken together, our results indicate that Cdc14 phosphatase functions in cell division and septum formation in F. graminearum, likely by counteracting Cdk phosphorylation, and is required for plant infection.

TaAbc1, a Member of Abc1-Like Family Involved in Hypersensitive Response against the Stripe Rust Fungal Pathogen in Wheat

To search for genes involved in wheat (Triticum aestivum L.) defense response to the infection of stripe rust pathogen Puccinia striiformis f. sp. tritici (Pst), we identified and cloned a new wheat gene similar to the genes in the Abc1-like gene family. The new gene, designated as TaAbc1, encodes a 717-amino acid, 80.35 kD protein. The TaAbc1 protein contains two conserved domains shared by Abc1-like proteins, two trans-membrane domains at the C-terminal, and a 36-amino acid chloroplast targeting presequence at the N-terminal. Characterization of TaAbc1 expression revealed that gene expression was tissue-specific and could be up-regulated by biotic agents (e.g., stripe rust pathogen) and/or by an abiotic stress like wounding. High-fold induction was associated with the hypersensitive response (HR) triggered only by avirulent stripe rust pathotypes, suggesting that TaAbc1 is a rust-pathotype specific HR-mediator. Down-regulating TaAbc1 reduced HR but not the overall resistance level in Suwon11 to CYR23, suggesting TaAbc1 was involved in HR against stripe rust, but overall host resistance is not HR-dependent.

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.