The DNA damage repair complex MoMMS21-MoSMC5 is required for infection-related development and pathogenicity of Magnaporthe oryzae

The conserved DNA damage repair complex,MMS21-SMC5/6(Methyl methane sulfonate 21-Structural maintenance of chromosomes 5/6),has been extensively studied in yeast,animals,and plants.However,its role in phytopathogenic fungi,particularly in the highly destructive rice blast fungus Magnaporthe oryzae,remains unknown.In this study,we functionally characterized the homologues of this complex,MoMMS21 and MoSMC5,in M.oryzae.We first demonstrated the importance of DNA damage repair in M.oryzae by showing that the DNA damage inducer phleomycin inhibited vegetative growth,infection-related development and pathogenicity in this fungus.Additionally,we discovered that MoMMS21 and MoSMC5 interacted in the nuclei,suggesting that they also function as a complex in M.oryzae.Gene deletion experiments revealed that both MoMMS21 and MoSMC5 are required for infection-related development and pathogenicity in M.oryzae,while only MoMMS21 deletion affected growth and sensitivity to phleomycin,indicating its specific involvement in DNA damage repair.Overall,our results provide insights into the roles of MoMMS21 and MoSMC5 in M.oryzae,highlighting their functions beyond DNA damage repair.

The Magnaporthe oryzae effector Avr-PikD suppresses rice immunity by inhibiting an LSD1-like transcriptional activator

Avirulence effectors(Avrs),encoded by plant pathogens,can be recognized by plants harboring the corresponding resistance proteins,thereby initiating effector-triggered immunity(ETI).In susceptible plants,however,Avrs can function as effectors,facilitating infection via effector-triggered susceptibility(ETS).Mechanisms of Avr-mediated ETS remain largely unexplored.Here we report that the Magnaporthe oryzae effector Avr-PikD enters rice cells via the canonical cytoplasmic secretion pathway and suppresses rice basal defense.Avr-PikD interacts with an LSD1-like transcriptional activator AKIP30 of rice,and AKIP30 is also a positive regulator of rice immunity,whereas Avr-PikD impedes its nuclear localization and suppresses its transcriptional activity.In summary,M.oryzae delivers Avr-PikD into rice cells to facilitate ETS by inhibiting AKIP30-mediated transcriptional regulation of immune response against M.oryzae.

培养过程参数对霉菌Rhizopus oryzae IFO细胞催化植物油脂合成生物柴油的影响研究

采用脂肪酶催化可再生动植物油脂合成生物柴油已经成为目前研究的热点,其中利用全细胞催化剂是一个重要的研究方向。文中直接利用霉菌R.oryzaeIFO细胞催化植物油脂甲醇解反应合成生物柴油,系统研究了培养过程参数对细胞生长和该细胞催化剂催化甲醇解反应活性的影响。研究表明,细胞培养过程中所添加的油脂不同,细胞在后续反应中催化特定油脂进行生物柴油制备时所表现出的催化活性也会有所差别;由某种油脂作为碳源得到的细胞催化剂催化对应油脂与甲醇转酯化反应制备生物柴油时,表现出比催化其他油脂和甲醇反应制备生物柴油更高的催化活性。在优化的操作参数(大豆精制油20g/L,蛋白胨70g/L,NaNO31.2g/L,KH2PO41.2g/L,MgSO4·7H2O0.5g/L,培养温度35℃,摇床转速130r/min)下,培养得到的细胞催化剂能有效催化大豆油与甲醇三步转酯化反应,生物柴油(脂肪酸甲酯)最终得率可达到86%。

固定化R.oryzae细胞发酵产胞内脂肪酶及其催化制备生物柴油

采用含胞内脂肪酶的微生物细胞催化油脂原料生产生物柴油是目前生物柴油生产领域的一个新的研究方向。探讨了以聚氨酯泡沫颗粒作为载体固定化R.oryzae细胞对细胞生长与产胞内脂肪酶的影响,并对固定化R.oryzae发酵条件进行了优化,特别对氮源的选用进行了研究。结果表明,选用廉价的豆粉作为有机氮源培养R.oryzae细胞效果较好,与以蛋白胨作为培养基氮源相比,单位培养基所得菌体的总酶活提高至1.55倍,同时,氮源利用率大大提高。进一步考察了有机氮源与无机氮源复合培养R.oryzae细胞的情况,发现豆粉与(NH4)2HPO4复合效果较好。利用正交设计对培养基中各种无机盐成分进行了优化。优化结果为全脂豆粉2%,大豆油3%,MgSO40.035%,K2HPO40.12%,(NH4)2HPO40.10%,上述培养条件下,单位培养基培养菌体胞内脂肪酶活可达6054.2U·L-1培养基。以该条件下培养菌体催化大豆油生产生物柴油,在叔丁醇体系下,反应24h生物柴油得率可达68.5%。

霉菌R.oryzae IFO催化大豆油脂合成生物柴油的研究

直接将霉菌R.oryzae IFO细胞应用于催化大豆油脂转酯合成生物柴油,并对其反应条件进行了优化.在反应所需甲醇分3次等量加入、醇油摩尔比1:1、反应温度35℃、缓冲液用量0.05mL/g油,缓冲液pH值范围3.6～6.98,生物柴油(脂肪酸甲酯)最终得率可达86%.

Isolation and Identification of Glyphosate-degraded Strain Aspergillus oryzae sp.A-F02 and Its Degradation Characteristics

[Objective] The research aimed to isolate the glyphosate-degraded strain and study its degradation characteristics.[Method] A glyphosate-degraded fungal strain A-F02 was isolated from sludge in an aeration tank of a glyphosate manufacture.The fungal strain A-F02 was identified according to morphological characteristics and internal transcribed spacer（ITS）region of nuclear ribosomal DNA sequence analysis.The glyphosate-biodegraded characteristics of strain A-F02 and the influencing factors were studied.[Result] The fungal strain A-F02 was identified as Aspergillus oryzae sp..The glyphosate-biodegraded rate was 86.82% in the mineral salt medium with 1 000 mg/L of glyphosate as the sole source of carbon,after being incubated at 30 ℃ and 150 rpm for 7 d.The biodegradation rates and biomass of the A-F02 were the highest under the culture conditions with glucose（0.5%,w/v）,pH 7.5,30 ℃ and glyphosate（1 500 mg/L）.[Conclusion] The research provided the experimental basis for glyphosate-biodegraded enzyme purification.

Enhancement of Rice Germplasm by Pyramiding the Cultivars with Multi-resistance to Orseolia oryzae (Wood-Mason)

[ Objective] This study was to breed rice cultivars with multi-resistance to Orseolia oryzae （Wood-Mason）. [ Method] The Guangxi local cultivar GX-M001 （Jiangchao） with high resistance to Orseolia oryzae （Wood-Mason） was used to hybrid with the known resistance cultivars ＂Kangwenqingzhan＂ （harboring GM5 gene）, OB677（ harboring GM3 gene） from Sri Lanka, HT1350 and high yield end quality cultivar ＂ Guiruanzhan＂. [ Result] Through pyramiding the multi-resistant genes via routine hybridization, the general resistances of the hybrids were remarkably enhanced. The grades of resistance were also improved, many of the combinations were endowed with a resistance at immune level （grade 0） ; and interestingly, the respective hybridization of GX-M001 （high resistance） with OB677（ medium resistance） and HT1350（suscepti- ble） also generate two lines at immune level, which is probably the effects of additive effects of genes.[ Conclusion] By routine hybridization, multiple genes were successfully pyramided, thus generating novel rice lines with multiple resistances. For the rice breeding scientists at the grass-roots level, the resistance-resistance pyramiding is an effective approach to breed high resistance cultivars.

固态共培养中丝状真菌对Aspergillus oryzae Su-16的影响

为了研究在固态基质中共培养的两种丝状真菌之间的相互影响,将Aspergillus oryzae Su-16与6株酒曲真菌分别混合接种麸皮培养基进行固态发酵,通过分析麸曲的α-淀粉酶活力、糖化酶活力、中性蛋白酶活力、纤维素酶活力以及麸曲的蛋白质含量、总酸、浸提液的pH值7个指标,借助SPSS16.0软件,利用Spearman相关系数分析,比较共培养麸曲与纯培养麸曲的加合之间的差异性。结合麸曲的SDS-PAGE的图谱分析和十字交叉培养法探讨A.oryzae Su-16在与6株真菌共培养体系中的相互影响的强弱。研究结果表明A.oryzae Su-16在与黑曲霉AN-13共培养中相互影响相对最大,且A.oryzae Su-16明显影响到黑曲霉AN-13的生理代谢而非生长分化,而微小毛霉RP-09则能显著影响到A.oryzae Su-16的分化和产孢。

叔丁醇介质体系中R.oryzae细胞催化大豆油制备生物柴油的研究

利用产胞内脂肪酶的微生物全细胞代替脂肪酶用于生物柴油的制备是降低酶法制备生物柴油成本的一条可行途径,近年来成为酶法制备生物柴油领域的研究热点。本论文探讨在叔丁醇介质体系下,以固定化R.oryzae全细胞作为催化剂催化大豆油制备生物柴油的可行性;考察叔丁醇用量、甲醇用量、缓冲液pH、初始含水量、菌体干重以及反应温度等因素对生物柴油得率的影响。在优化反应条件下,以大豆油为原料,反应24h生物柴油得率可达70%左右。比较了固定化R.oryzae全细胞在叔丁醇介质体系与无溶剂体系两种体系下的稳定性。结果表明,叔丁醇介质体系下R.oryzae全细胞回用稳定性显著提高。

水稻条斑病细菌(Xanthomolias oryzae pv.oryzicola)Wzt基因参与LPS O-抗原合成和影响细菌致病性

ABC-转运系统将同质O-抗原多糖链从细胞质内膜转运到细胞周质空间合成脂多糖(LPS)。通过功能互补和亚克隆序列分析在X. oryzae pv. oryzicola的基因组文库中发现了一个Wzt基因, 该基因编码产物是运输O-抗原的ABC-转运系统的疏水组成部分,为ATP-结合蛋白。为区别基因来源将该基因命名为Wzt_(Xooc). Wzt_(Xooc)编码一个35.9 ku的蛋白质。通过分析发现,Wzt_(Xooc)与数据库中的其他细菌包括水稻白叶枯病菌的ABC-转运系统的ATP结合蛋白质不同。在Wzt_(Xooc)序列中仅发现ATP-结合蛋白中4个保守基序的3个,没有发现ATP-结合位点Walker A(ATP/GTP binding site motif A)。通过基因插入突变得到Wzt_(Xooc)基因突变体Mwzt。LPS分析表明:由于该基因突变使O- 抗原链不能转运通过细胞质膜,不能形成完整的LPS分子,突变体菌落表面丧失了产生大量胞外多糖的能力;突变细菌不产生鞭毛,丧失了游动性和生物膜形成的能力。重要的是突变体在水稻上的繁殖能力和致病性明显下降,证明Wzt_(Xooc)基因与LPS合成及致病性有关。

Aspergillus oryzae IF-39蛋白酶水解玉米醇溶蛋白

Aspergillus oryzae IF-39固态发酵生产蛋白酶,利用该蛋白酶水解经碱处理的玉米醇溶蛋白,以蛋白的水解度为指标确定最佳酶解条件。结果表明:底物质量浓度10%、加酶量7500U/g、pH7.0、反应温度30℃、反应时间2h,在此最佳条件下水解度可达到33.5%。

Aspergillus oryzae HN3.042孢子增殖培养基的统计优化(英文)

运用Plackett–Burman和均一设计(UD)对影响Aspergillus oryzae HN3.042孢子增殖的无机盐进行筛选和优化。在筛选的11种无机盐中,KH2PO4,(NH4)2SO4,MgSO4和FeCl3对孢子繁殖有显著正效应。在此基础上,运用UD设计进行添加量优化,孢子增殖最大程度时的无机盐组成为:KH2PO4,4.57g/kg;(NH4)2SO4,1.96g/kg;MgSO4,1.72g/kg和FeCl3,0.64g/kg。在此条件下,孢子数达到1.07×1011个/g干曲,较未优化前提高了66.13%;同时,曲中的中性蛋白酶活力也提高了14.39%。

Formation Conditions of Aspergiilus oryzae Protoplast

[Objective] To explore the efficient way for the preparation of Aspergillus oryzae protoplast.[Method] The statistics of protoplast yield by the enzymolysis of mycelia with different culture time,different osmotic stabilizers and different concentrations of enzyme system was carried out in this study.[Result] The protoplast yield was higher when the mycelium was cultivated for 108 h,and the osmotic stabilizer was 0.8 mol/L of NaCl.With the enzymes system of 2 mg/ml of lysozyme,6 mg/ml of cellulose and 6 mg/ml of glusulase,the protoplast yield achieved its highest,which was up to 9.0×105 per ml.[Conclusion] The yield of protoplast was affected by the situation of mycelia themselves and external conditions.This study had provided a basis for the preparation of a large number of active protoplasts and the further researches on cell fusion.

Population Genetic Structure of Magnaporthe oryzae in Rice Blast Epidemic Areas in Hubei Province

Single-spore isolates were obtained from rice-growing fields of Yuan＇an in Hubei Province where rice blast seriously occurs in some years. DNA fingerprints were divided into 112 haplotypes and 14 lineages at 73% genetic similarity level. Among the lineages, no dominant lineages were found. The population genetic structures of Magnaporthe oryzae were not distinctly different in different years. The analysis also showed that there wasn＇t obvious simple relationship between patho- types and fingerprint groups.

新型固定化Aspergillus oryzae脂肪酶催化合成1,3-二油酸-2-棕榈酸甘油三酯

旨在评价自制固定化sn-1,3专一性脂肪酶Aspergillus oryzae L03(AOL)替代商业固定化脂肪酶酸解法合成1,3-二油酸-2-棕榈酸甘油三酯(1,3-dioleoyl-2-palmitoylglycerol,OPO)的可行性。以棕榈硬脂和油酸为原料,在正己烷体系中,得到AOL最适反应参数为:反应温度55℃,反应时间1 h,棕榈硬脂和油酸的底物摩尔比为1∶8,正己烷与底物混合物质量比1∶6,AOL水分含量3.5%(w/w)。该条件下,OPO含量44.9%,三棕榈酸甘油酯(PPP)含量为3.23%,sn-2位棕榈酸占所有棕榈酸比例为68.86%,符合国家标准。同时,AOL和LipozymeRM IM、LipozymeTL IM两种商品酶对比,AOL的催化速率最快,1 h达到最大OPO生产峰值。经过8次循环使用,AOL具有良好的操作稳定性。研究表明,自制的新型廉价固定化酶AOL具有良好的应用前景。

制固态曲参数对酱油曲中Aspergillus oryzae NCFEC03产蛋白酶活力的影响

以5种米曲霉为出发菌株,筛选获得产蛋白酶活力最强的米曲霉Aspergillus oryzae NCFEC03,通过单因素试验研究麸皮豆粕比、加水量、接种量、时间和培养温度等酱油固态制曲因素对Aspergillus oryzaeNCFEC03产蛋白酶的影响。选取影响较大的3个因素:麸皮豆粕比、加水量和接种量进行正交试验。经极差和方差分析发现,对酱油曲中蛋白酶活力影响的先后顺序为:麸皮豆粕比>加水量>接种量,其中麸皮豆粕比具有最显著的影响。最佳制曲条件为麸皮豆粕比1∶1、加水量80%(V/W,干曲)、接种量0.8%、30℃条件下培养48h,米曲霉Aspergillus oryzae NCFEC03产蛋白酶活力为(1687.6±4.9)U/g干曲。

The arms race between Magnaporthe oryzae and rice: Diversity and interaction of Avr and R genes

Rice blast disease, caused by Magnaporthe oryzae, threatens global food security. The rice blast pathosystem is a longstanding model system for understanding plant-microbe interactions. In order to elucidate the coevolution of the host and pathogen, and provide the appropriate methods for preventing or controlling rice blast disease, researchers have focused on the evolution of virulence factors and resistance genes. Thus far, more than 30 rice blast resistance（R） genes and 12 avirulence（Avr） genes have been cloned. This review summarizes the cloned rice blast R genes, cloned Avr genes of M. oryzae and the interaction between them. This discussion also considers some of the major unanswered questions concerning this pathosystem and the opportunities for future investigations.

Optimization of Solid-State Fermentation with Lactobacillus brevis and Aspergillus oryzae for Trypsin Inhibitor Degradation in Soybean Meal

The aim of the present study was to optimize trypsin inhibitor degradation in soybean meal by solid-state fermentation （SSF） with Lactobacillus brevis and Aspergillus oryzae, and to determine the effect of SSF on phytic acid, crude protein, crude fat, and amino acid profile. Response surface methodology （RSM） with Box-Behnken design was used to optimize SSF. The optimal conditions derived from RSM for L. brevis fermentation were： pH=5. 1; inoculum size=10%; duration=72 h; substrate to water ratio=1.5. The minimum content of trypsin inhibitors was 6.4 mg g^-1 dry matter. The optimal conditions derived from RSM for A. oryzae fermentation were： substrate to water ratio= 0.8 1; inoculum size=4%; duration=120 h. The minimum content of trypsin inhibitors was 1.6 mg g^-1 dry matter. Both L. brevis and A. oryzae decreased trypsin inhibitors dramatically （57.1 and 89.2% respectively）. L. brevis fermentation did not affect phytic acid （0.4%） and crude fat （5.2%） considerably, whereas A. oryzae fermentation degraded phytic acid （34.8%） and crude fat （22.0%） contents to a certain extent. Crude protein content was increased after both fermentation （6.4 and 12.9% for L. brevis and A. oryzae respectively）. Urease activity was reduced greatly （83.3 and 58.3% for L. brevis and A. oryzae respectively）. In conclusion, SSF with A. oryzae and L. brevis reduced trypsin inhibitor content and modified major macronutrients in soybean meal.

Functional Analysis of MYB73 of Arabidopsis thaliana Against Bipolaris oryzae

Bipolaris oryzae is a severe rice disease,resulting in significant economic losses.MYB73 gene of Arabidopsis was isolated based on T-DNA insertion mutants whose lose-of-function mutant increased susceptibility to B.oryzae.Results of staining for H2O2 revealed that infection of incompatible B.oryzae caused much more strong brown patches on the leaves of myb73 mutant than those on the leaves of wild type.Expression of MYB73 gene was induced by B.oryzae.Its increased expression was severely impaired in the myb73 mutant.Expression of MYB73 was severely decreased in the npr1,jar1,eds5,and sid2 mutants,suggesting MYB73 gene participates in the jasmonate （JA） and salicylic acid （SA） signaling pathways.Expression of MYB73 who encoded an R2R3 MYB transcription factor was increased by SA,JA,and ethylene （ET） treatments.The cDNA full-length sequence of MYB73 was 960 bp and a protein with 320 amino acids was encoded.The predicted molecular weight of MYB73 was 34.85 kDa.The expression of MYB73 gene was induced within 24 h of the inoculation,however,the expression of PR1,PDF1.2 and NPR1 weren＇t changed.When B.oryzae successfully infected myb73 mutant plants,the expression of PR1,PDF1.2 and NPR1 were increased.Collectively,our results suggested that MYB73 is involved in NPR1-mediated SA and JA signaling pathways.

A Non-Marker Mutagenesis Strategy to Generate Poly-hrp Gene Mutants in the Rice Pathogen Xanthomonas oryzae pv. oryzicola

Xanthomonas oryzae pv.oryzicola （Xoc）,the critical pathogen causing bacterial leaf streak in rice,possesses a hrp cluster that is responsible for triggering hypersensitive response （HR） in non-host tobacco and pathogenicity in host rice,and is considered to be one of the model pathogens in the rice model plant.Here,we developed a high-throughput mutagenesis system using a two-step integration mediated by a novel suicide vector pKMS1.It was used to generate single or poly-gene mutants of hpa1,hpa2,hrcV,hrpE,hpaB,and hrpF gene for functional analysis.In total,five single,four double,and two triple hrp gene mutants were constructed.The double and triple hrp gene deletion mutants triggered novel phenotypes in planta.Our data suggest that pKMS1 is a useful tool for non-marker mutagenesis of multiple genes in Xoc.