Comparative Nuclear Proteomics Analysis Provides Insight into the Mechanism of Signaling and Immune Response to Blast Disease Caused by Magnaporthe oryzae in Rice

Modulation of plant immune system by extrinsic/intrinsic factors and host‐specific determinants fine‐tunes cellular components involving multiple organelles, particularly nucleus to mount resistance against pathogen attack. Rice blast, caused by hemibiotrophic fungus Magnaporthe oryzae, is one of the most devastating diseases that adversely affect rice productivity. However, the role of nuclear proteins and their regulation in response to M. oryzae remains unknown. Here, the nucleus‐associated immune pathways in blast‐resistant rice genotype are elucidated. Temporal analysis of nuclear proteome is carried out using 2‐DE coupled MS/MS analysis. A total of 140 immune responsive proteins are identified associated with nuclear reorganization, cell division, energy production/deprivation, signaling, and gene regulation. The proteome data are interrogated using correlation network analysis that identified significant functional modules pointing toward immune‐related coinciding processes through a common mechanism of remodeling and homeostasis. Novel clues regarding blast resistance include nucleus‐associated redox homeostasis and glycolytic enzyme–mediated chromatin organization which manipulates cell division and immunity. Taken together, the study herein provides evidence that the coordination of nuclear function and reprogramming of host translational machinery regulate resistance mechanism against blast disease.     

Proteomic analysis of salicylic acid-induced resistance to Magnaporthe oryzae in susceptible and resistant rice

To probe salicylic acid (SA)-induced sequential events at translational level and factors associated with SA response, we conducted virulence assays and proteomic profiling analysis on rice resistant and susceptible cultivars against Magnaporthe oryzae at various time points after SA treatment. The results showed that SA significantly enhanced rice resistance against M. oryzae. Proteomic analysis of SA-treated leaves unveiled 36 differentially expressed proteins implicated in various functions, including defense, antioxidative enzymes, and signal transduction. Majority of these proteins were induced except three antioxidative enzymes, which were negatively regulated by SA. Consistent with the above findings, SA increased the level of reactive oxygen species (ROS) with resistant cultivar C101LAC showing faster response to SA and producing higher level of ROS than susceptible cultivar CO39. Furthermore, we showed that nucleoside diphosphate kinase 1, which is implicated in regulation of ROS production, was strongly induced in C101LAC but not in CO39. Taken together, the findings suggest that resistant rice cultivar might possess a more sensitive SA signaling system or effective pathway than susceptible cultivar. In addition, our results indicate that SA also coordinates other cellular activities such as photosynthesis and metabolism to facilitate defense response and recovery, highlighting the complexity of SA-induced resistance mechanisms.     

稻瘟菌Magnaporthe oryzae P-ATPases基因家族分析

利用TCDB(Transporter Classification Database)网站数据库中的P-ATPases氨基酸序列对稻瘟菌全基因组表达序列(Coding Sequence,CDS)数据库进行搜索和分析,共发现23个P-ATPases基因,进化树分析表明这23个基因分属于4个家族和7个亚家族。构建了本地ESTs数据库,通过P-ATPases基因CDS序列与EST序列比对分析发现,在这些基因中有20个存在EST同源体,另外3个基因没有发现EST同源体,因此这20个基因是真实P-ATPases基因的可能性更高。运用MEME程序分析了这些P-ATPases蛋白结构域的基序,有6种基序在90%以上的基因氨基酸序列中出现,属保守基序。对这23个P-ATPases基因GC含量的分析表明,它们的平均GC含量在0.519-0.628之间,稍高于稻瘟菌整个基因组GC平均含量(0.516),同时这些基因内各区段GC含量变化不大,没有明显的梯度变化。本文结果为下一步深入研究稻瘟菌中P-ATPases基因家族的功能奠定了基础。     

稻瘟病菌分泌蛋白MoCDIE2诱导植物细胞死亡的功能分析

由稻瘟病菌(Magnaporthe oryzae)引起的稻瘟病是全球最严重的植物真菌病害之一。稻瘟病菌通过分泌效应蛋白进入与植物相互作用界面或转运到植物细胞内,抑制寄主植物的免疫防卫反应,使病原菌成功侵染。通过农杆菌介导的异源表达策略,筛选到能引起非寄主植物烟草细胞死亡的候选效应蛋白MoCDIE2(Cell Death-Inducing Effector)。序列分析表明:MoCDIE2基因编码一个蓖麻毒素B凝集素蛋白;系统发育树构建结果表明MoCDIE2同源蛋白保守存在于丝状真菌中;利用基因敲除的方法获得MoCDIE2的敲除突变体,结果表明MoCDIE2的敲除突变体在菌丝生长和致病性方面与野生型菌株Guy11没有明显差异。     

Rice varieties with resistance to multiple races of Magnaporthe oryzae offer opportunities to manage rice blast in Australia

Rice blast caused by Magnaporthe oryzae is the most destructive disease of rice worldwide. Development of resistant varieties is considered as the most cost‐effective and sustainable way to manage rice blast. However, there remains a lack of knowledge about the resistance of rice varieties to blast disease in Australia. This study was conducted to determine if there was any resistance existing among the rice varieties grown in Australia to M. oryzae isolates from this country that belong to different races. There was a resistant reaction of the variety SHZ‐2 to all the five races of IA‐1, IA‐3, IA‐63, IB‐3 and IB‐59, with a percent disease index (%DI) less than 40. Varieties NTR587, BR‐IRGA‐409, Ceysvoni and Rikuto Norin 20 showed a resistant reaction to races IA‐3, IA‐63, IB‐3 and IB‐59; and the variety Kyeema exhibited a resistant reaction to races IA‐3, IB‐3 and IB‐59. For the races IA‐1 and IB‐59 with more than one isolate, varieties with differential disease reactions across different isolates belonging to the same race were also revealed: five varieties, Langi, Opus, Sherpa, Viet 1 and Topaz, exhibited differential disease reactions to the three IA‐1 isolates; 10 varieties showed differential disease reactions to the four IB‐59 isolates; in addition, the varieties that had differential disease reactions to the IA‐1 isolates also exhibited differential disease reactions to the IB‐59 isolates of race. This study provides valuable resistance sources for breeding programmes to develop rice varieties with resistance to multiple races of M. oryzae in Australia.     

Twinfilin regulates actin assembly and Hexagonal peroxisome 1 (Hex1) localization in the pathogenesis of rice blast fungus Magnaporthe oryzae

Actin assembly at the hyphal tip is key for polar growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. The mechanism of its precise assemblies and biological functions is not understood. Here, we characterized the role of M. oryzae Twinfilin (MoTwf) in M. oryzae infection through organizing the actin cables that connect to Spitzenkörper (Spk) at the hyphal tip. MoTwf could bind and bundle the actin filaments. It formed a complex with Myosin2 (MoMyo2) and the Woronin body protein Hexagonal peroxisome 1 (MoHex1). Enrichment of MoMyo2 and MoHex1 in the hyphal apical region was disrupted in a ΔMotwf loss-of-function mutant, which also showed a decrease in the number and width of actin cables. These findings indicate that MoTwf participates in the virulence of M. oryzae by organizing Spk-connected actin filaments and regulating MoHex1 distribution at the hyphal tip.     

Inheritance of dsRNAs in the rice blast fungus, Magnaporthe grisea

The 1.6 and 1.8 kbp dsRNAs have been found in the rice blast fungus, Magnaporthe grisea strain MG01. These dsRNA molecules are located in cytoplasm of the fungal cells and maintained stably during vegetative growth. Three crosses between dsRNA free and dsRNA containing strains including a parental cross, sib-mating and back cross were made to follow the inheritance of dsRNAs during sexual reproduction. Approximately 10% of ascospore progenies (11 out of 105) contained dsRNAs from all three crosses. These data indicate that dsRNAs of M. grisea are inherited at a low frequency and not in a Mendelian fashion.     

稻瘟菌WD重复功能域中SSR的变异及其对蛋白结构的影响

含WD重复功能域的蛋白能够参与信号传导、转录调控、RNA剪切、细胞的凋亡等多种功能,在病原菌与寄主植物蛋白互作的过程中扮演着重要的角色。本研究分析了稻瘟病菌基因组中94个WD功能域基因编码区和调控区中SSR的组成、分布,并检测了7个蛋白编码区中SSR的变异及其对蛋白二级结构的影响。结果表明,WD功能域基因的编码区和调控区中都含有大量的SSR,但是SSR在这些基因的外显子区、内含子区、5’一UTR和3’一UTR区中SSR的组成和分布均不相同;编码区中三碱基和六碱基SSR分布较多,这些SSR基序大都表现为GC含量较高和其所编码的亲水性氨基酸出现的频率远远高于疏水性氨基酸的特点。且检测的7个WD功能域基因的编码区中的SSR位点均具有丰富的多态性,通过Antheprot（DPM）软件预测发现：SSR的变异对蛋白的二级结构有一定影响。这暗示着SSR的变异对致病相关基因的变异起着十分重要的作用。     

转录调控因子OxyRxoo对水稻白叶枯病菌H2O2降解途径的调控作用

摘要：【目的】为了阐明水稻白叶枯病菌(Xanthomonas oryzae pv. oryzae, 简称Xoo)转录调控因子OxyRxoo对过氧化氢(H2O2) 降解途径的调控作用。【方法】本研究对推导的H2O2识别调控基因oxyRxoo进行了基因克隆、序列分析、缺失突变和互补试验及其相关表型的鉴定。【结果】克隆的oxyRxoo基因序列与其它几种病原黄单胞菌的同源序列高度保守。OxyRxoo是LysR家族成员之一,具有PBPb结构域和DNA结合保守结构域(HTH)。用标记交换法构建了△oxyRxoo基因缺失突变体。与野生型菌株PXO99A相比,尽管△oxyRxoo在离体培养条件下的生长无明显变化,但H2O2抗性显著地降低,过氧化物酶(CAT)活性明显下降,基因互补可以使之恢复; 过氧化物酶基因表达下调, oxyRxoo自身表达显著上调。【结论】OxyRxoo作为一个重要转录调控因子,调控了Xoo的 H2O2降解途径。     

Pex14/17, a filamentous fungus‐specific peroxin,is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.     

Serotonin attenuates biotic stress and leads to lesion browning caused by a hypersensitive response to Magnaporthe oryzae penetration in rice

The hypersensitive response (HR) of plants is one of the earliest responses to prevent pathogen invasion. A brown dot lesion on a leaf is visual evidence of the HR against the blast fungus Magnaporthe oryzae in rice, but tracking the browning process has been difficult. In this study, we induced the HR in rice cultivars harboring the blast resistance gene Pit by inoculation of an incompatible M. oryzae strain, which generated a unique resistance lesion with a brown ring (halo) around the brown fungal penetration site. Inoculation analysis using a plant harboring Pit but lacking an enzyme that catalyzes tryptamine to serotonin showed that high accumulation of the oxidized form of serotonin was the cause of the browning at the halo and penetration site. Our analysis of the halo browning process in the rice leaf revealed that abscisic acid enhanced biosynthesis of serotonin under light conditions, and serotonin changed to the oxidized form via hydrogen peroxide produced by light. The dramatic increase in serotonin, which has a high antioxidant activity, suppressed leaf damage outside the halo, blocked expansion of the browning area and attenuated inhibition of plant growth. These results suggest that serotonin helps to reduce biotic stress in the plant by acting as a scavenger of oxygen radicals to protect uninfected tissues from oxidative damage caused by the HR. The deposition of its oxide at the HR lesion is observed as lesion browning.