Effects of MEK-Specific Inhibitor U0126 on the Conidial Germination,Appressorium Production,and Pathogenicity of Setosphaeria turcica

Systemic studies on the effects of mitogen-activated protein kinase （MAPK） signal transduction pathway on the growth and development of Setosphaeria turcica is helpful not only in understanding the molecular mechanism of pathogenhost interaction but also in the effective control of the diseases caused by S. turcica. U0126, the specific MEK inhibitor, is used to treat S. turcica before the observation of the conidial germination, appressorium production, and pathogenicity of the pathogen. There is no significant effect of U0126 on the colony morphology and mycelium growth of the pathogen. After treatment with U0126, the growth of mycelium and conidia are normal, but the conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are significantly inhibited. Under the definite concentration scope, an increase in U0126 concentration increases the inhibition degree of conidial germination and appressorium production, but the inhibition degree decreases with elongation of treatment time. The conidial germination, appressorium production, and pathogenicity of S. turcica on susceptible corn leaves are regulated by the MAPK pathway inhibited by U0126.

Representative appressorium stage cDNA library of Magnaporthe grisea

A mature appressorium cDNA library of rice blast fungus, Magnaporthe grisea, was constructed in a λTriplEx2 vector by SMART?cDNA library containing 2.37×106 independent clones about 100% of which harbor foreign cDNA inserts with average size of 660 bp. Of 9 randomly selected clones, 2 expressed sequence tags (ESTs) sequences did not have homologous EST sequences of M grisea in GenBank. The appressorium cDNA library is suitable for gene expression analysis and function analysis of the late stages of appressorium formation and the early stages of penetration of M grisea.

Investigation of the biological roles of autophagy in appressorium morphogenesis in Magnaporthe oryzae

Magnaporthe oryzae has been used as a primary model organism for investigating fungus-plant interaction. Many researches focused on molecular mechanisms of appressorium formation to restrain this fungal pathogen. Autophagy is a very high conserved process in eukaryotic cells. Recently, autophagy has been considered as a key process in development and differentia-tion in M. oryzae. In this report, we present and discuss the current state of our knowledge on gene expression in appressorium formation and the progress in autophagy of rice blast fungi.

Melanin,DNA replication,and autophagy affect appressorium development in Setosphaeria turcica by regulating glycerol accumulation and metabolism

Setosphaeria turcica(syn.Exserohilum turcicum)is the pathogenic fungus of maize(Zea mays)that causes northern leaf blight,which is a major maize disease worldwide.Melanized appressoria are highly specialized infection structures formed by germinated conidia of S.turcica that infect maize leaves.The appressorium penetrates the plant cuticle by generating turgor,and glycerol is known to be the main source of the turgor.Here,the infection position penetrated by the appressorium on maize leaves was investigated,most of the germinated conidia entered the leaf interior by directly penetrating the epidermal cells,and the appressorium structure was necessary for the infection,whether it occurred through epidermal cells or stomata.Then,to investigate the effects of key factors in the development of the appressorium,we studied the effects of three inhibitors,including a melanin inhibitor(tricyclazole,TCZ),a DNA replication inhibitor(hydroxyurea,HU),and an autophagy inhibitor(3-methyladenine,3-MA),on appressorium turgor and glycerol content.As results,appressorium turgor pressure and glycerol concentration in the appressorium reached their highest levels at the mature stage of the appressorium under the control and inhibitor treatments.The three inhibitors had the greatest effects on appressorium turgor pressure at this stage.Glycogen and liposomes are the main substances producing glycerol.It was also found inhibitors affected the distribution of glycogen and liposomes,which were detected in the conidia,the germ tube,and the appressorium during appressorium development.This study provides profound insight into the relationship between appressorium turgor pressure and glycerol content,which was affected by the synthesis of melanin,DNA replication,and autophagy in the developing appressorium during a S.turcica infection.

Germination and appressorium formation of Pyricularia oryzae Cavara can be inhibited by reduced concentration of Blasin~? Flowable with carbon dioxide microbubbles

We investigated the possibility to reduce the usage of Blasin^Flowable （BF）, a disinfectant inhibiting the germination and appressorium formation of Pyricularia oryzae Cavara conidia, by using carbon dioxide microbubbles （CO2MB）. Germination was significantly inhibited by 10 000-fold diluted BF solution containing CO2MB generated by the decompression-type generator compared to CO2 millibubbles （CO2MMB） and CO2MB generated by the gas-water circulating-type generator. Appressodum formation in the 10 000-fold diluted BF solution containing both CO2MBs was less than that in CO2MMB. Scanning electron microscopy showed wrinkles and dents on the surface of conidia treated with 5 000-fold diluted BF solution containing both CO2MBs. Via transmission electron microscopy, we observed the expansion of the vacuole and the intracellular space and bloated or absent lipid granules in the conidia treated with BF solution containing both CO2MBs. Our results show that inhibition of the conidium germination and appressorium formation of P. oryzae Cavara by 10 000-fold diluted BF solution could be achieved by using the decompression-type CO2MB.

A lipid droplet-associated protein Nem1 regulates appressorium function for infection of Magnaporthe oryzae

Lipid droplets are important storages in fungal conidia and can be used by plant pathogenic fungi for infection.However,the regulatory mechanism of lipid droplets formation and the utilization during fungal development and infection are largely unknown.Here,in Magnaporthe oryzae,we identified a lipid droplet-associated protein Nem1 that played a key role in lipid droplets biogenesis and utilization.Nem1 was highly expressed in conidia,but lowly expressed in appressoria,and its encoded protein was localized to lipid droplets.Deletion of NEM1 resulted in reduced numbers of lipid droplets and decreased content of diacylglycerol(DAG)or triacylglycerol(TAG).NEM1 was required for asexual development especially conidia production.TheΔnem1 mutant was nearly loss of virulence to host plants due to defects in appressorial penetration and invasive growth.Remarkably,Nem1 was regulated by the TOR signaling pathway and involved in the autophagy process.The Ser303 residue of Nem1 could be phosphorylated by the cAMP-PKA signaling pathway and was important for biological function of Nem1.Together,our study revealed a regulatory mechanism of lipid biogenesis and metabolism during the conidium and appressorium formation of the rice blast fungus.

解淀粉芽胞杆菌YN-J3对禾生炭疽菌孢子萌发和附着胞形成的影响

燕麦炭疽病是由禾生炭疽菌Colletotrichumcereale引起的作物病害,可以危害植株地上部。为研究解淀粉芽胞杆菌Bacillus amyloliquefaciens YN-J3对禾生炭疽菌的抑菌作用机理,用菌株YN-J3代谢液、菌体悬浮液、代谢物处理和培养皿对扣熏蒸处理禾生炭疽菌分生孢子,并对孢子萌发率和附着胞形成率进行测定。结果表明,经菌株YN-J3代谢液直接处理的禾生炭疽菌分生孢子形成的不正常附着胞体积变大,没有黑色素沉积。透射电镜观察发现,禾生炭疽菌不正常附着胞的细胞壁疏松、质膜崩溃、细胞器解体、细胞质空泡化。YN-J3菌株菌体悬浮液抑制禾生炭疽菌分生孢子萌发、附着胞形成的EC50值为1.14×10^(5)、1.91×10^(4) cfu/mL。将YN-J3菌株菌体悬浮液和禾生炭疽菌分生孢子用0.22μm滤膜隔开培养,禾生炭疽菌分生孢子萌发和附着胞形成比例明显下降,10~8 cfu/mL菌体浓度下30 h形成不正常附着胞比例为15.67%。菌体代谢液的乙酸乙酯抽提液能够完全抑制分生孢子萌发和附着胞的形成。与菌株YN-J3菌体对扣培养30 h的分生孢子不萌发。以上结果表明,解淀粉芽胞杆菌YN-J3菌株的代谢物,能够抑制禾生炭疽菌分生孢子萌发和附着胞形成并产生一种不正常附着胞,菌株YN-J3抑制禾生炭疽菌侵染结构的形成。

Identification of a key G-protein coupled receptor in mediating appressorium formation and fungal virulence against insects

Fungal G-protein coupled receptors(GPCRs)play essential roles in sensing environmental cues including host signals.The study of GPCR in mediating fungus-insect interactions is still limited.Here we report the evolution of GPCR genes encoded in the entomopathogenic Metarhizium species and found the expansion of Pth11-like GPCRs in the generalist species with a wide host range.By deletion of ten candidate genes MrGpr1–MrGpr10 selected from the six obtained subfamilies in the generalist M.robertsii,we found that each of them played a varied level of roles in mediating appressorium formation.In particular,deletion of MrGpr8 resulted in the failure of appressorium formation on different substrates and the loss of virulence during topical infection of insects but not during injection assays when compared with the wild-type(WT)strain.Further analysis revealed that disruption of MrGpr8 substantially impaired the nucleus translocation of the mitogen-activated protein kinase(MAPK)Mero-Fus3 but not the MAPK Mero-Slt2 during appressorium formation.We also found that the defect ofΔMrGpr8 could not be rescued with the addition of cyclic AMP for appressorium formation.Relative to the WT,differential expression of the selected genes have also been detected inΔMrGpr8.The results of this study may benefit the understanding of fungus-interactions mediated by GPCRs.

Transcriptional regulation of secondary metabolism and autophagy genes in response to DNA replication stress in Setosphaeria turcica

The fungal pathogen Setosphaeria turcica causes northern corn leaf blight(NCLB),which leads to considerable crop losses.Setosphaeria turcica elaborates a specialized infection structures called appressorium for maize infection.Previously,we demonstrated that the S.turcica triggers an S-phase checkpoint and ATR(Ataxia Telangiectasia and Rad3 related)-dependent self-protective response to DNA genotoxic insults during maize infection.However,how the regulatory mechanism works was still largely unknown.Here,we report a genome wide transcriptional profile analysis during appressorium formation in the present of DNA replication stress.We performed RNA-Seq analysis to identify S.tuicica genes responsive to DNA replication stress.In the current work,we found that appressorium-mediated maize infection by S.turcica is significantly blocked by S-phase checkpoint.A large serial of secondary metabolite and melanin biosynthesis genes were blocked in appressorium formation of S.turcica during the replication stress.The secondary metabolite biosynthesis genes including alcohol dehydrogenase GroES-like domain,multicopper oxidase,ABCtransporter families,cytochrome P450 and FAD-containing monooxygenase were related to plant pathogen infection.In addition,we demonstrated that autophagy in S.turcica is up-regulated by ATR as a defense response to stress.We identified StATG3,StATG4,StATG5,StATG7 and StATG16 genes for autophagy were induced by ATR-mediated S-phase checkpoint.We therefore propose that in response to genotoxic stress,S.turcica utilizes ATR-dependent pathway to turn off transcription of genes governing appressorium-mediated infection,and meanwhile inducing transcription of autophagy genes likely as a mechanism of self-protection,aside from the more conservative responses in eukaryotes.

不同诱导因素对玉米大斑病菌附着胞产生的影响

附着胞是许多叶部病原真菌的重要侵染机构。在不同温度、光照条件、基质和培养时间下,对玉米大斑病菌1号小种和2号小种分别在玻璃平板和玉米叶片上进行附着胞的室内诱导,发现分生孢子的水悬浮液在玻璃平板和玉米叶片的上表面及下表面都可产生附着胞,附着胞都能正常萌发并产生侵入丝,但在玉米叶片上附着胞和侵入丝的产生时间要晚于玻璃平板,产生数量也少。玉米大斑病菌分生孢子在PD培养基中产生的附着胞数目少于清水处理。影响玉米大斑病菌附着胞生长和发育的决定因素是温度,在玻璃平板上10～36℃培养24h后就可以产生附着胞,但最适宜的温度是18～25℃,低于15℃和高于30℃都不利于附着胞的产生。不同菌株在同一玉米品种的叶片上产生的附着胞数量不同,但是同一菌株在抗病品种和感病品种间产生的附着胞数量差异不显著。

稻瘟病菌单克隆抗体的研制及其对稻瘟病菌附着胞形成的影响

稻温病菌的分生孢子、芽管、附着胞的混合物作为抗原免疫BALB/c小鼠 ,取免疫小鼠的脾细胞与SP2 /0骨髓瘤细胞在 50 %PEG下融合成杂交瘤细胞 ,用间接ELISA筛选阳性孔 ,获 1 1株单克隆抗体。间接免疫荧光试验表明其中 4株单克隆抗体 2B4、4A1 、1D1 和 2H4分别与孢子、芽管或附着胞有特异性结合 ;Westernblotting分析发现 2B4、4A1 、1D1 单克隆抗体分别与孢子、芽管表面的提取物有不同的结合带 ;此四株单克隆抗体均干扰稻温病菌附着胞形成 ,并抑制稻温病菌在叶表的致病性。

几个炭疽菌株的分类鉴定

作者对８个炭疽菌株分离物进行鉴定，明确了核桃、枣、山茱萸、棉花和柑桔寄主上５个分离菌株为胶孢炭疽菌［Ｃｏｌｌｅｔｏｔｒｉｃｈｕｍｇｌｏｅｏｓｐｏｒｉｏｉｄｅｓ（Ｐｅｎｚ．）Ｓａｃｃ．］；大豆寄主上分离菌株为黑线炭疽菌［Ｃ．ｄｅｍａｔｉｕｍ（Ｐｅｒｓ．ｅｘＦｒ．）Ｇｒｏｖｅ］；辣椒寄主上分离菌株为辣椒炭疽菌［Ｃ．ｃａｐｓｉｃｉ（Ｓｙｄ．）Ｂｕｔｌ．ｅｔＢｉｓｂｙ］；西瓜寄主上分离物为瓜类炭疽菌［Ｃ．ｏｒｂｉｃｕｌａｒｅ（ＢｅｒｋｅｔＭｏｎｔ．）Ａｒｘ．］。

果蔬采后病害的潜伏侵染机理研究进展

潜伏侵染是引起果蔬采后病害的重要因素,病原菌侵染途径多样、侵染时期难以预测,采后处理不能完全控制果蔬潜伏侵染病害发生。潜伏侵染性病原菌附着胞的形成及分泌的致病因子如胞外酶、活性氧(reactive oxygen species,ROS)、p H因子和毒素是病原菌穿透寄主细胞的重要原因,同时寄主组织衰老防卫能力降低也加速了病原菌在寄主细胞内次生菌丝的形成和寄主病症的出现。本文通过对果蔬采后病害的潜伏侵染机理的综述,明确病原菌侵入寄主的机制及病原菌在寄主内长期潜伏的原因,可以有针对性地防治采后病害,对寻找防治潜伏侵染新方法和提高采后病害的防治效率具有理论意义。

植物病原真菌黑色素与致病性关系的研究进展

黑色素是一类生物聚合分子的总称,不同来源的黑色素种类不同,其中报道较多的是DOPA黑色素和DHN黑色素。DOPA黑色素和DHN黑色素具有相似的理化性质但其合成底物和途径不同。DHN黑色素在植物病原真菌中广泛存在,与病原菌致病能力密切相关。病原菌侵染寄主时黑色素沉积在附着胞细胞壁的内层,防止了形成膨压的溶质渗透到细胞外,产生很大的机械压力,保证病原菌侵入寄主。结合作者的研究结果综述了黑色素的种类、性质及黑色素与病原菌致病性关系等方面的研究进展。

枯草芽孢杆菌菌株A抗菌蛋白的分离纯化及抗真菌机理

【目的】揭示生防菌枯草芽孢杆菌(Bacillus subtilis)菌株A产生的抗真菌蛋白的性质及作用机理。【方法】采用硫酸铵沉淀菌株A发酵液获粗蛋白;采用DE52离子交换层析技术对粗蛋白进行分离纯化;对具抗真菌活性的纯化组分采用MALDI-TOF-MS进行结构分析;以玉米小斑病菌(Bipolaris maydis)为材料,对纯化活性蛋白的抗真菌机理进行研究。【结果】从粗蛋白中分离到一种抗真菌活性蛋白(FV),与RNA结合蛋白Hfq Bacillus subtilis subsp.subtilis gi|16078797的匹配率为86.484%,相对分子质量为8 508.5 Da,等电点为8.04。FV对玉米小斑病菌孢子萌发的半数抑制浓度(IC50)为138.32μg.mL-1,附着胞形成的IC50为22.23μg.mL-1。经FV处理后,玉米小斑病菌菌丝顶端、中部出现膨大泡囊,呈"念珠状";扫描电镜观察发现泡囊状菌丝表面凹凸不平,并残留有碎片;透射电镜观察发现在部分菌丝细胞原生质出现"空泡"的菌丝里有"子菌丝"。【结论】枯草芽孢杆菌菌株A产生的抗真菌蛋白与RNA结合蛋白Hfq高度同源,可抑制致病真菌孢子萌发、附着胞形成,并影响菌丝细胞的形态和结构。

枯草芽孢杆菌C-D6抗菌蛋白对稻瘟病菌的抑菌作用

枯草芽孢杆菌Bacillus subtilis菌株C-D6及其发酵滤液在平板上对稻瘟病菌Magnaporthe oryzae显示明显的抑制作用。通过20%饱和度硫酸铵沉淀和Sephadex G-75凝胶柱层析分离,获得1个影响病原菌附着胞形成的抗菌蛋白,SDS-聚丙烯酰胺凝胶电泳检测发现该蛋白的分子量为32kD。显微观察发现:用该蛋白处理的孢子虽能完全萌发,但形成串珠状膨大的芽管和色浅、膨大的附着胞,并抑制芽管的正常生长。该蛋白的活性表现具有较好的热稳定性(80℃,30min)和较广的pH适应范围(5.6～9.3)。

玉米大斑病菌黑色素的一些理化性质和光谱吸收特征

黑色素是某些植物和动物真菌病害的致病相关因子,不同来源的黑色素其生物合成途径可能不同。对玉米大斑病菌细胞壁结合黑色素和从培养滤液中提取的黑色素进行理化性质、紫外吸收光谱和红外光谱扫描测定,并与标准品黑色素进行比较分析,明确了玉米大斑病菌黑色素具有与标准品黑色素相似的理化性质。DHN黑色素的特异性抑制剂——三环唑,对玉米大斑病菌0号和1号小种黑色素的产生均有抑制作用;以玉米大斑病菌基因组DNA为模板,通过PCR扩增,得到了1,3,8-三羟基萘还原酶基因的同源片段,推测玉米大斑病菌黑色素合成于DHN途径。

DHN黑色素与玉米大斑病菌附着胞膨压形成的关系

【目的】探讨玉米大斑病菌胞内DHN黑色素在附着胞膨压产生过程中的作用,明确玉米大斑病菌的侵入机理。【方法】通过诱导玉米大斑病菌附着胞产生,确定附着胞形成的最佳条件,利用溶质排斥技术及incipient-cytorrhysis技术对玉米大斑病菌野生型菌株01-23和黑色素缺失突变体△St3hnr附着胞细胞壁孔径大小和膨压进行测定。【结果】野生型菌株01-23细胞壁孔径范围是2.1—2.7 nm,膨压在5.4 MPa左右;黑色素缺失突变体△St3hnr细胞壁孔径范围是2.7—3.3 nm,膨压在4.1 MPa左右;缺乏黑色素的附着胞不能形成高膨压,丧失了穿透能力。【结论】黑色素层对溶质分子外渗的阻挡作用导致了附着胞高膨压的产生,膨压产生的机械穿透力在玉米大斑病菌穿透基质平面的过程中发挥了重要作用。

2A型蛋白磷酸酶对玉米大斑病菌菌体发育的调控作用

【目的】明确2A型蛋白磷酸酶(PP2A)在玉米大斑病菌致病过程中的作用,为研发新型杀真菌制剂和探讨植物病害防治新策略奠定理论基础。【方法】利用不同浓度的PP2A特异性抑制剂——斑蝥素(cantharidin)处理玉米大斑病菌,研究在该抑制剂作用下玉米大斑病菌的菌落生长、分生孢子产量、孢子萌发、附着胞发育、黑色素合成及HT-毒素活性。【结果】随着斑蝥素浓度的增加,其对菌丝体生长的抑制作用也逐渐增强,当达到160μmol.L-1时,培养8 d的菌落平均直径仅为对照的41.7%;同时,处理组产孢量均高于对照组,160μmol.L-1斑蝥素处理组产孢量达到了对照组的15.5倍。分析斑蝥素对病菌分生孢子的萌发、附着胞形成及侵染的影响表明,上述3个阶段对斑蝥素的敏感性由强到弱依次为附着胞形成>分生孢子萌发>附着胞侵染;此外,对照组的胞内黑色素含量为0.13 g.L-1,处理组的胞内黑色素含量均高于对照组,且随着斑蝥素浓度的增加不断升高。【结论】PP2A特异性抑制剂斑蝥素对玉米大斑病菌菌落生长、附着胞发育具有抑制作用,而对分生孢子产生及胞内黑色素的合成具有一定的促进作用。

西双版纳海巴戟炭疽病和病原菌分生孢子萌发、附着胞形成条件的研究

对西双版纳海巴戟炭疽病进行调查和病原菌鉴定。结果表明，海巴戟炭疽病周年发生，8月的发病率和病情指数最高，分别为40．76％和8．9。根据病原菌形态特征，将其鉴定为瓜类炭疽菌[Colletotrichum orbiculare（Berk．＆Mont）]。有关温度、pH、光照和碳源对病原菌孢子萌发和附着胞形成影响的研究显示，20～35℃，海巴戟炭疽病菌分生孢子萌发率高，20℃是形成附着胞的最适温度；最适合海巴戟炭疽病菌分生孢子萌发和形成附着胞的pH为4～8；在日光灯连续光照、12h光暗交替和自然光照射条件下分生孢子萌发率在90％以上，光照对此病原菌附着胞形成的影响差异不大；1％葡萄糖、麦芽糖、蔗糖、D-果糖和α-乳糖溶液中孢子萌发率都不高。