水稻免疫机制研究进展

植物先天免疫主要由两部分组成：一类是通过细胞膜上的病原菌分子模式识别受体识别病原微生物表面存在的分子特征激发的免疫反应（PTI）;另一类是专化性的抗病R蛋白识别病原微生物的效应蛋白,从而激发下游的病原菌小种特异性的防卫反应过程（ETI）．随着水稻抗病信号途径中越来越多的抗病基因以及关键的调控基因被克隆和功能鉴定,同时多种水稻病原菌效应蛋白的发现,水稻抗病机理的研究也越来越深入．本文阐述了水稻的PTI,ETI及其下游参与免疫信号转导的关键性组分,从而形成一个初步的水稻免疫调控网络．     

水稻抗稻瘟病天然免疫机制及抗病育种新策略

稻瘟病是水稻最严重的病害之一,由子囊菌(Magnaporthe oryzae)引起。利用抗病品种是防治稻瘟病最经济、最有效的措施。近年来,稻瘟病已发展为研究植物与病原真菌分子互作机制的模式系统,在水稻与稻瘟菌互作和寄主抗性分子生物学、基因组学和蛋白组学等领域取得了一系列重要的研究成果。文章综述了近年来水稻抗稻瘟病两种天然免疫机制,即病原菌相关分子模式诱导和效应蛋白诱导的抗病机制研究的最新进展,讨论了GWAS、TALLEN、CRISPR和HIGS等基因组研究新方法和新技术在水稻抗病育种中的应用,并对目前稻瘟病抗性机制研究和抗病育种中的问题和挑战进行了探讨和展望。     

A Rapid and Simple Method for DNA Preparation of Magnaporthe oryzae from Single Rice Blast Lesions for PCR-Based Molecular Analysis

Rice blast is one of the most destructive diseases of rice worldwide, and the causative agent is the filamentous ascomycete Magnaporthe oryzae. With the successful cloning of more and more avirulence genes from M. oryzae, the direct extraction of M. oryzae genomic DNA from infected rice tissue would be useful alternative for rapid monitoring of changes of avirulence genes without isolation and cultivation of the pathogen. In this study, a fast, low-cost and reliable method for DNA preparation of M. oryzae from a small piece of infected single rice leaf or neck lesion was established. This single step method only required 10 min for DNA preparation and conventional chemical reagents commonly found in the laboratory. The AvrPik and AvrPi9 genes were successfully amplified with the prepared DNA. The expected DNA fragments from 570 bp to 1,139 bp could be amplified even three months after DNA preparation. This method was also suitable for DNA preparation from M. oryzae strains stored on the filter paper. All together these results indicate that the DNA preparation method established in this study is reliable, and could meet the basic needs for polymerase chain reaction-based analysis of M. oryzae.     

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。     

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.     

稻瘟病菌无毒基因研究进展

水稻与稻瘟病菌之间的特异互作符合基因对基因假说。本文将从稻瘟病菌与水稻抗病基因间的互作特点、稻瘟病菌的分子标记、已克隆的稻瘟病菌无毒基因三个方面对稻瘟病菌无毒基因研究进展作简要介绍     

Rice ubiquitin-conjugating enzyme OsUBC26 is essential for immunity to the blast fungus Magnaporthe oryzae

The functions of ubiquitin-conjugating enzymes (E2) in plant immunity are not well understood. In this study, OsUBC26, a rice ubiquitin-conjugating enzyme, was characterized in the defence against Magnaporthe oryzae. The expression of OsUBC26 was induced by M. oryzae inoculation and methyl jasmonate treatment. Both RNA interference lines and CRISPR/Cas9 null mutants of OsUBC26 reduced rice resistance to M. oryzae. WRKY45 was down-regulated in OsUBC26 null mutants. In vitro E2 activity assay indicated that OsUBC26 is an active ubiquitin-conjugating enzyme. Yeast two-hybrid assays using OsUBC26 as bait identified the RING-type E3 ligase UCIP2 as an interacting protein. Coimmunoprecipitation assays confirmed the interaction between OsUBC26 and UCIP2. The CRISPR/Cas9 mutants of UCIP2 also showed compromised resistance to M. oryzae. Yeast two-hybrid screening using UCIP2 as bait revealed that APIP6 is a binding partner of UCIP2. Moreover, OsUBC26 working with APIP6 ubiquitinateds AvrPiz-t, an avirulence effector of M. oryzae, and OsUBC26 null mutation impaired the proteasome degradation of AvrPiz-t in rice cells. In summary, OsUBC26 plays important roles in rice disease resistance by regulating WRKY45 expression and working with E3 ligases such as APIP6 to counteract the effector protein AvrPiz-t from M. oryzae.     

内生细菌B10对稻瘟病菌抑制作用的初步研究

目的:研究内生细菌B10对水稻稻瘟病菌的抑制作用,为菌株的应用提供理论依据。方法:采用菌丝生长速率法、孢子萌发法和田间试验测定内生细菌发酵上清液和菌液对稻瘟病菌的抑制作用。结果:内生细菌B10发酵上清液对稻瘟病菌菌丝生长和孢子萌发有较强的抑制作用,100倍稀释液对菌丝生长的抑制率为79.37%,对孢子萌发的抑制率为63.42%,对稻瘟病的田间防治效果达70.2%以上。结论:内生细菌B10对稻瘟病有较强的抑菌作用。     

Magnaporthe oryzae nucleoside diphosphate kinase is required for metabolic homeostasis and redox-mediated host innate immunity suppression

The fungus Magnaporthe oryzae causes blast, the most devastating disease of cultivated rice. After penetrating the leaf cuticle, M. oryzae grows as a biotroph in intimate contact with living rice epidermal cells before necrotic lesions develop. Biotrophic growth requires maintaining metabolic homeostasis while suppressing plant defenses, but the metabolic connections and requirements involved are largely unknown. Here, we characterized the M. oryzae nucleoside diphosphate kinase-encoding gene NDK1 and discovered it was essential for facilitating biotrophic growth by suppressing the host oxidative burst—the first line of plant defense. NDK enzymes reversibly transfer phosphate groups from tri- to diphosphate nucleosides. Correspondingly, intracellular nucleotide pools were perturbed in M. oryzae strains lacking NDK1 through targeted gene deletion, compared to WT. This affected metabolic homeostasis: TCA, purine and pyrimidine intermediates, and oxidized NADP⁺, accumulated in Δndk1. cAMP and glutathione were depleted. ROS accumulated in Δndk1 hyphae. Functional appressoria developed on rice leaf sheath surfaces, but Δndk1 invasive hyphal growth was restricted and redox homeostasis was perturbed, resulting in unsuppressed host oxidative bursts that triggered immunity. We conclude Ndk1 modulates intracellular nucleotide pools to maintain redox balance via metabolic homeostasis, thus quenching the host oxidative burst and suppressing rice innate immunity during biotrophy.     

Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Abstract

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.     

2,6‐Dimethoxy‐1,4‐Benzoquinone Enhances Resistance Against the Rice Blast Fungus Magnaporthe oryzae

We investigated the effect of 2,6‐dimethoxy‐1,4‐benzoquinone (DMBQ) on induced resistance to Magnaporthe oryzae in rice. DMBQ concentrations greater than 50 μg/ml inhibited spore germination and appressorium formation in M. oryzae. When rice leaves pretreated with 10 μg/ml DMBQ, which did not show antifungal activity against spore germination and appressorium formation of M. oryzae, were inoculated with M. oryzae spores 5 days after DMBQ pretreatment, blast lesion formation was inhibited compared with control leaves pretreated with distilled water. In addition, infection‐inhibiting activity against M. oryzae was significantly enhanced in rice leaf sheaths pretreated with 10 μg/ml DMBQ. H₂O₂ generation was observed in rice leaves pretreated with DMBQ, and PAL, POX, CHS and PR10a were significantly expressed in these leaves. These results suggested that DMBQ can protect rice from blast disease caused by M. oryzae.     

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.     

稻瘟病菌侵染诱导的水稻早期反应基因的cDNA片段克隆与序列分析

以稻瘟病菌感染水稻,利用ｍＲＮＡ 差异显示技术分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１（ｅａｒｌｙ ｒｅｓｐｏｎｓｉｖｅ ｇｅｎｅ） 的ｃＤＮＡ 片段。Ｎｏｒｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因在稻瘟病菌侵染水稻叶片６ ｈ 后开始表达,８ ｈ 最强,１０ ～１２ ｈ 开始减弱,１６ ｈ 消失。Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分析表明,ＥＲ１ 基因属于水稻基因组。对ＥＲ１ 基因片段（２１９ ｂｐ） 进行了克隆和序列分析。经查询,在ＧｅｎＢａｎｋ 中没有与ＥＲ１ 同源的基因序列。     

SOBIR1 and AGB1 independently contribute to nonhost resistance to Pyricularia oryzae (syn. Magnaporthe oryzae) in Arabidopsis thaliana

ABSTRACT

Rice blast caused by Pyricularia oryzae (syn. Magnaporthe oryzae) is a disease devastating to rice. We have studied the Arabidopsis-P. oryzae pathosystem as a model system for nonhost resistance (NHR) and found that SOBIR1, but not BAK1, is a positive regulator of NHR to P. oryzae in Arabidopsis. AGB1 is also involved in NHR. However, the genetic interactions between SOBIR1, BAK1, and AGB1 are uncharacterized. In this study, we delineated the genetic interactions between SOBIR1, BAK1, and AGB1 in NHR to P. oryzae in Arabidopsis and found SOBIR1 and AGB1 independently control NHR to P. oryzae in Arabidopsis pen2-1 mutant plants. Furthermore, XLG2, but not TMM, has a positive role in penetration resistance to P. oryzae in Arabidopsis pen2-1 mutant plants. Our study characterized genetic interactions in Arabidopsis NHR.

Abbreviations: PRR: pattern recognition receptor, RLK: receptor-like kinase, RLP: receptor-like protein, BAK1: BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED RECEPTOR KINASE 1, BIR1: BAK1-INTERACTING RECEPTOR-LIKE KINASE 1, SOBIR1: SUPPRESSOR OF BIR1-1-1, AGB1: ARABIDOPSIS G PROTEIN ß-SUBUNIT 1, XLG2: EXTRA-LARGE G PROTEIN 2     

Crystal Structure of Manganese Lipoxygenase of the Rice Blast Fungus Magnaporthe oryzae

Lipoxygenases (LOX) are non-heme metal enzymes, which oxidize polyunsaturated fatty acids to hydroperoxides. All LOX belong to the same gene family, and they are widely distributed. LOX of animals, plants, and prokaryotes contain iron as the catalytic metal, whereas fungi express LOX with iron or with manganese. Little is known about metal selection by LOX and the adjustment of the redox potentials of their protein-bound catalytic metals. Thirteen three-dimensional structures of animal, plant, and prokaryotic FeLOX are available, but none of MnLOX. The MnLOX of the most important plant pathogen, the rice blast fungus Magnaporthe oryzae (Mo), was expressed in Pichia pastoris. Mo-MnLOX was deglycosylated, purified to homogeneity, and subjected to crystal screening and x-ray diffraction. The structure was solved by sulfur and manganese single wavelength anomalous dispersion to a resolution of 2.0 Å. The manganese coordinating sphere is similar to iron ligands of coral 8R-LOX and soybean LOX-1 but is not overlapping. The Asn-473 is positioned on a short loop (Asn-Gln-Gly-Glu-Pro) instead of an α-helix and forms hydrogen bonds with Gln-281. Comparison with FeLOX suggests that Phe-332 and Phe-525 might contribute to the unique suprafacial hydrogen abstraction and oxygenation mechanism of Mo-MnLOX by controlling oxygen access to the pentadiene radical. Modeling suggests that Arg-525 is positioned close to Arg-182 of 8R-LOX, and both residues likely tether the carboxylate group of the substrate. An oxygen channel could not be identified. We conclude that Mo-MnLOX illustrates a partly unique variation of the structural theme of FeLOX.