小麦叶锈菌休眠与萌发夏孢子的差异表达

【目的】小麦叶锈菌引起的小麦叶锈病是小麦的重要病害之一,孢子萌发和侵入气孔是保证叶锈菌正常侵染寄主的重要前提。本研究旨在研究小麦叶锈菌夏孢子萌发的差异表达特性,为揭示萌发的机制及其与致病的关系提供依据。【方法】利用小麦叶锈菌致病类型THTT的休眠夏孢子和萌发夏孢子进行RNA-seq分析。【结果】在萌发夏孢子中筛选出相比休眠夏孢子上调表达的基因为4400个,下调基因5325个。GO富集分析发现,上调差异基因主要涉及细胞进程、有机物代谢、信号传导、单个有机体过程、催化酶活性等;下调基因主要涉及单组织过程、单个有机体细胞过程、有机物代谢过程、催化活性调节等过程。利用KEGG分析发现,差异基因共参与109条通路,从中筛选出两条与孢子萌发相关的通路——丝裂原活化蛋白激酶(MAPK)信号通路和囊泡运输中的SNARE蛋白交流。10个基因的定量分析结果与基因数字表达谱分析结果一致。【结论】研究获得了叶锈菌孢子萌发及侵入气孔过程中的重要差异基因,研究结果为研究叶锈菌致病机制奠定基础。     

小麦隐匿柄锈菌与小麦互作不同阶段的基因差异表达分析

[目的]小麦叶锈病是影响小麦生产主要病害之一,其病原菌新小种的出现和劣势小种上升为优势小种导致抗病品种的抗病性不断被克服.小麦隐匿柄锈菌与小麦互作不同阶段差异表达谱分析对于揭示该病菌致病的分子机制,进而有效防控小麦叶锈病具有重要意义.[方法]利用转录组分析小麦隐匿柄锈菌致病生理小种与感叶锈病小麦品种MuTcLr19亲和...     

Reactive Oxygen Species Play a Role in the Infection of the Necrotrophic Fungi,Rhizoctonia solani in Wheat

Rhizoctonia solani is a nectrotrophic fungal pathogen that causes billions of dollars of damage to agriculture worldwide and infects a broad host range including wheat, rice, potato and legumes. In this study we identify wheat genes that are differentially expressed in response to the R. solani isolate, AG8, using microarray technology. A significant number of wheat genes identified in this screen were involved in reactive oxygen species (ROS) production and redox regulation. Levels of ROS species were increased in wheat root tissue following R. solani infection as determined by Nitro Blue Tetrazolium (NBT), 3,3''-diaminobenzidine (DAB) and titanium sulphate measurements. Pathogen/ROS related genes from R. solani were also tested for expression patterns upon wheat infection. TmpL, a R. solani gene homologous to a gene associated with ROS regulation in Alternaria brassicicola, and OAH, a R. solani gene homologous to oxaloacetate acetylhydrolase which has been shown to produce oxalic acid in Sclerotinia sclerotiorum, were highly induced in R. solani when infecting wheat. We speculate that the interplay between the wheat and R. solani ROS generating proteins may be important for determining the outcome of the wheat/R. solani interaction.     

The spatiotemporal control of KatG2 catalase-peroxidase contributes to the invasiveness of Fusarium graminearum in host plants

Reactive oxygen species (ROS) are involved in the pathogen-host interactions, and play a Janus-faced role in the resistance and susceptibility of plants to biotrophic and necrotrophic pathogens. The ascomycete fungus Fusarium graminearum causes hazardous wheat Fusarium head blight worldwide. Deletion of the putative secreted catalase-peroxidase gene in F. graminearum, KatG2, reduced the virulence in wheat spike infection. However, it remains unclear when and where KatG2 scavenges ROS during the invasion of wheat. In this study, we delineate the change in ROS levels in the transition of the infection phase under microscopic observation. Correspondingly, the pathogen switches its strategy of infection with temporal and spatial regulation of KatG2 to counteract oxidative stress generated by host plant cells. With the native promoter-driven KatG2-mRFP strain, we show that KatG2-mRFP expression was induced in planta and accumulated in the infection front region at the early infection stage. In contrast to its ubiquitous cellular localization in runner hyphae, KatG2-mRFP is exclusively located on the cell wall of invading hyphal cells, especially at the pathogen-host cellular interface. Using posttranslational modification analysis, we found that asparagine residues at the 238 and 391 positions of KatG2 could be modified by N-glycosylation and that these two residues are required for KatG2 accumulation and cell wall localization in planta.     

Phenotypic and genetic diversity of leaf rust resistance in wheat wild relatives

Puccinia triticina (Pt), the causal agent of leaf rust evolves through forming new pathotypes that adversely affect the growth and yield of wheat cultivars. Therefore, continued production of resistant varieties through exploring novel sources of resistance in wild relatives which are abundantly found in Iran and the neighbouring regions is a major task in wheat breeding programs. The aim of the present study was to explore 60 wild wheat genotypes selected from the species Triticum monococcum, Aegilops tauschii, Ae. neglecta, Ae. cylindrica, Ae. triuncialis, Ae. umbellulata, Ae. speltoides, Ae. columnaris, Ae. crassa and Ae. ventricosa for resistance to leaf rust. The cultivar ‘Boolani’ and Thatcher near-isogenic lines were used as controls. Two-week-old seedlings were inoculated using 10 Pt pathotypes, and the infection types were recorded. The genotypes were also analysed for polymorphism using six sequence-tagged sites (STS) and sequence characterized amplified region (SCAR) markers. Forty-eight genotypes produced high infection types (3⁺) for two pathotypes, but the remaining genotypes produced low infection types of ‘0; ^=’ to ‘1⁺CN’ to all pathotypes. The latter included three accessions of Ae. tauschii, two accessions of each Ae. umbellulata, Ae. columnaris and Triticum monococcum, and one accession from each Ae. triuncialis, Ae. ventricosa and Ae. neglecta. Analysis for STS and SCAR markers suggested several genotypes could carry the genes Lr9, Lr10, Lr19, Lr24, Lr26 and Lr37 or their potential orthologs in addition to unknown resistance genes. In conclusion, the identified resistant genotypes could be further characterized and used in wheat breeding programs for leaf rust resistance.     

Tolerance to oxidative stress is required for maximal xylem colonization by the xylem‐limited bacterial phytopathogen,Xylella fastidiosa

Bacterial plant pathogens often encounter reactive oxygen species (ROS) during host invasion. In foliar bacterial pathogens, multiple regulatory proteins are involved in the sensing of oxidative stress and the activation of the expression of antioxidant genes. However, it is unclear whether xylem‐limited bacteria, such as Xylella fastidiosa, experience oxidative stress during the colonization of plants. Examination of the X. fastidiosa genome uncovered only one homologue of oxidative stress regulatory proteins, OxyR. Here, a knockout mutation in the X. fastidiosa oxyR gene was constructed; the resulting strain was significantly more sensitive to hydrogen peroxide (H₂O₂) relative to the wild‐type. In addition, during early stages of grapevine infection, the survival rate was 1000‐fold lower for the oxyR mutant than for the wild‐type. This supports the hypothesis that grapevine xylem represents an oxidative environment and that X. fastidiosa must overcome this challenge to achieve maximal xylem colonization. Finally, the oxyR mutant exhibited reduced surface attachment and cell–cell aggregation and was defective in biofilm maturation, suggesting that ROS could be a potential environmental cue stimulating biofilm development during the early stages of host colonization.     

Flaxseed Oil for Control of Peanut Rust and Its Disease Control Mechanism

Severity of peanut rust caused by Puccinia arachidis was reduced by 15 edible oils tested. Flaxseed oil was the best suppressing the disease completely. Peanut oil, wheat germ oil, brown rice oil, aloe oil, olive oil and corn germ oil also caused more than 75% reduction in disease incidence. Flaxseed oil reduced the rust to a negligible level in the greenhouse and was nearly as effective as the fungicide chlorothalonil in peanut field trials. The control of peanut rust by flaxseed oil did not result from activation of the host defence mechanisms. Flaxseed oil did not affect urediniospore germination, but reduced the germ tube length and completely suppressed appressorium formation which is essential for the pathogen to form an infection peg to pass through the stomatal aperture and infect the host tissue. Although the pathogen had penetrated, flaxseed oil still exerted some inhibitory effect against the growth of the pathogen. The advantages of using flaxseed oil to control peanut rust are that it is relatively inexpensive, easy to prepare, and friendly to the environment and human health.     

Identification of a hydrogen peroxide signalling pathway in the control of light-dependent germination in Arabidopsis

Germination is controlled by external factors, such as temperature, water, light and by hormone balance. Recently, reactive oxygen species (ROS) have been shown to act as messengers during plant development, stress responses and programmed cell death. We analyzed the role of ROS during germination and demonstrated that ROS in addition to their role as cell wall loosening factor are essential signalling molecules in this process. Indeed, we showed that ROS are released prior to endosperm rupture, that their production is required for germination, and that class III peroxidases, as ROS level regulators, colocalized with ROS production. Among ROS, H₂O₂ modifies, during germination early steps, the expression of genes encoding for enzymes regulating ROS levels. This pointing out a regulatory feedback loop for ROS production. Measurements of endogenous levels of ROS following application of GA and ABA suggested that ABA inhibits germination by repressing ROS accumulation, and that, conversely, GA triggers germination by promoting an increase of ROS levels. We followed the early visible steps of germination (testa and endosperm rupture) in Arabidopsis seeds treated by specific ROS scavengers and as the light quality perception is necessary for a regular germination, we examined the germination in presence of exogenous H₂O₂ in different light qualities. H₂O₂ either promoted germination or repressed germination depending on the light wavelengths, showing that H₂O₂ acts as a signal molecule regulating germination in a light-dependent manner. Using photoreceptors null-mutants and GA-deficient mutants, we showed that H₂O₂-dependent promotion of germination relies on phytochrome signalling, but not on cryptochrome signalling, and that ROS signalling requires GA signalling.     

Antifungal Activity of Aqueous Extracts of the Leaves of Cowparsnip and Comfrey

We found that extracts from the leaves of medicinal comfrey and cowparsnip strongly inhibit the germination of Erysiphe graminisconidia and uredospores of Puccinia graminis. Spraying wheat seedlings with these extracts, in contrast to the irrigation of soil, markedly diminished infection in plants with powdery mildew. Antifungal activity in vitroand protective activity (when plants were sprayed) correlated with the level of phenolic compounds in these extracts. Experiments with healthy plants have demonstrated that the photosynthetic apparatus of wheat plants is stimulated by extracts. Spraying seedlings with the extracts resulted in an increased rate of O₂evolution calculated per unit of chlorophyll, an increase in the ratio (F_M– F_T)/F_Tin the experiments that recorded slow fluorescence induction, an increase in the relative light intensity of band A, and a decrease of relative intensity of band Cin experiments with thermoluminescence of wheat leaves. These results provide evidence that the protective activity of comfrey and cowparsnip extracts is associated with their action on the pathogenic fungus and with the activation of natural defense reactions of the host plant.     

Microarray analysis of the gene expression profile in the midgut of silkworm infected with cytoplasmic polyhedrosis virus

In order to obtain an overall view on silkworm response to Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) infection, a microarray system comprising 22,987 oligonucluotide 70-mer probes was employed to compare differentially expressed genes in the midguts of BmCPV-infected and normal silkworm larvae. At 72 h post-inoculation, 258 genes exhibited at least 2.0-fold differences in expression level. Out of these, 135 genes were up-regulated, while 123 genes were down-regulated. According to gene ontology (GO), 140 genes were classified into GO categories. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis indicates that 35 genes were involved in 10 significant (P < 0.05) KEGG pathways. The expressions of genes related to valine, leucine, and isoleucine degradation, retinol metabolism, and vitamin B6 metabolism were all down-regulated. The expressions of genes involved in ribosome and proteasome pathway were all up-regulated. Quantitative real-time polymerase chain reaction was performed to validate the expression patterns of 13 selected genes of interest. The results suggest that BmCPV infection resulted in the disturbance of protein and amino acid metabolism and a series of major physiological and pathological changes in silkworm. Our results provide new insights into the molecular mechanism of BmCPV infection and host cell response.     

Optimization of the HyPer sensor for robust real‐time detection of hydrogen peroxide in the rice blast fungus

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant‐pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H₂O₂) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H₂O₂ dynamics in real time during important stages of the plant infection process.