Apoptosis-related genes confer resistance to Fusarium wilt in transgenic 'Lady Finger' bananas

Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is one of the most devastating diseases of banana (Musa spp.). Apart from resistant cultivars, there are no effective control measures for the disease. We investigated whether the transgenic expression of apoptosis-inhibition-related genes in banana could be used to confer disease resistance. Embryogenic cell suspensions of the banana cultivar, 'Lady Finger', were stably transformed with animal genes that negatively regulate apoptosis, namely Bcl-xL, Ced-9 and Bcl-2 3' UTR, and independently transformed plant lines were regenerated for testing. Following a 12-week exposure to Foc race 1 in small-plant glasshouse bioassays, seven transgenic lines (2 × Bcl-xL, 3 × Ced-9 and 2 × Bcl-2 3' UTR) showed significantly less internal and external disease symptoms than the wild-type susceptible 'Lady Finger' banana plants used as positive controls. Of these, one Bcl-2 3' UTR line showed resistance that was equivalent to that of wild-type Cavendish bananas that were included as resistant negative controls. Further, the resistance of this line continued for 23-week postinoculation at which time the experiment was terminated. Using TUNEL assays, Foc race 1 was shown to induce apoptosis-like features in the roots of wild-type 'Lady Finger' plants consistent with a necrotrophic phase in the life cycle of this pathogen. This was further supported by the observed reduction in these effects in the roots of the resistant Bcl-2 3' UTR-transgenic line. This is the first report on the generation of transgenic banana plants with resistance to Fusarium wilt.     

香蕉枯萎病菌Fow1基因的克隆及序列分析

为了解Fow1基因在尖镰刀菌古巴专化型侵染香蕉过程中的作用,及其与尖镰刀菌古巴专化型生理小种1号和生理小种4号之间的致病力差异的关系,采用PCR和RT-PCR方法扩增了2个生理小种的Fow1基因,并对扩增产物进行了克隆测序及相似序列搜索和比对,还对基因编码的蛋白进行了结构预测和功能分析。研究结果表明2个生理小种Fow1基因开放阅读框均为957bp,编码318个氨基酸,基因序列和氨基酸序列差异小,而且两个生理小种Fow1基因所编码的蛋白均具有酵母线粒体载体蛋白典型的结构特征,推测Fow1基因可能为香蕉枯萎病菌在香蕉组织中定殖所必需。从Fow1基因序列及其编码蛋白的氨基酸序列看,2个生理小种致病力的差异与Fow1基因并无明显对应关系,这为进一步研究Fow1基因功能奠定了基础。     

香蕉枯萎病菌Argonaute基因功能分析及其调控小RNA发生

尖孢镰刀菌古巴专化型Fusarium oxysporum f.sp.cubense是威胁香蕉生产的重要土传病原真菌,其中4号生理小种(Foc4)能感染几乎所有的栽培品系.Argonaute蛋白(AGO)介导的RISC复合体在RNAi干扰中起到重要作用.Foc4含有两个进化上高度保守的AGO蛋白,本研究利用同源重组技术获...     

Differential Colonization Patterns of Bananas (Musa spp.) by Physiological Race 1 and Race 4 Isolates of Fusarium oxysporum f.sp. cubense

Fusarium oxysporum f.sp. cubense (Foc) is the causative agent of Fusarium wilt of bananas (Musa spp.). To clarify the colonization patterns of Foc in bananas, two green fluorescent protein‐tagged isolates, NT320 (race 1) and B2‐gfp (race 4), were used to follow infection of the banana varieties Pisang Awak and Brazil. Penetration and colonization of both isolates in roots of these two banana varieties were observed within 6 days, but sporulation in xylem vessels was not observed until day 30 postinoculation. Interestingly, B2‐gfp penetrated into xylem vessels of Pisang Awak banana roots more quickly than NT320, implying that the race 4 isolate is more virulent than the race 1 isolate. This result was further confirmed by comparing the disease severity of plants inoculated with NT320 with that of plants inoculated with B2‐gfp. Quantitative real‐time PCR revealed that some pathogenicity‐associated genes, including Fga1, Fhk1, Fow2 and Ste12, were upregulated by B2‐gfp during exposure to Brazil bananas, while they were either downregulated by NT320 or not significantly changed. These data might partly explain why the race 4 isolate was more virulent than the race 1 isolate.     

The effector SIX8 is required for virulence of Fusarium oxysporum f.sp. cubense tropical race 4 to Cavendish banana

Plant pathogens employ effectors as molecular weapons to manipulate host immunity and facilitate colonization. Fusarium oxysporum f. sp. cubense is the agent of wilt disease in banana plantlets and four races of the pathogen have been identified based on the cultivar specificity. A total of 9 SIX genes have been detected in the genome of Foc TR4 and 6 genes detected in Foc1. Among these SIX genes, SIX2 and SIX8 are only detected in Foc TR4, not identified in Foc1. Expression profiles analysis revealed that SIX genes of Foc TR4 are highly induced after inoculation to Cavendish banana plantlets. Virulence analysis of the SIX2 and SIX8 knock-out mutants showed that SIX8 is required for the virulence of Foc TR4 while SIX2 has no obvious functions. Over expression of SIX8-FLAG proteins in the SIX8 knock-out mutant partly restored the virulence. Western blot analysis suggested that SIX8 could be secreted into the extracellular space and a signal peptide resided the N-terminal polypeptide sequence. This study provides some clues for further research on mechanism of SIX8 in regulating virulence of Foc TR4.     

A proteomic study of in-root interactions between chickpea pathogens: the root-knot nematode Meloidogyne artiellia and the soil-borne fungus Fusarium oxysporum f. sp. ciceris race 5

Fusarium wilt caused by the fungus Fusarium oxysporum f. sp. ciceris (Foc) is the main soil-borne disease limiting chickpea production. Management of this disease is achieved mainly by the use of resistant cultivars. However, co-infection of a Foc-resistant plant by the fungus and the root-knot nematode Meloidogyne artiellia (Ma) causes breakdown of the resistance and thus limits its efficacy in the control of Fusarium wilt. In this work we aimed to reveal key aspects of chickpea:Foc:Ma interactions, studying fungal- and nematode-induced changes in root proteins, using chickpea lines 'CA 336.14.3.0' and 'ICC 14216K' that show similar resistant (Foc race 5) and susceptible (Ma) responses to either pathogen alone but a differential response after co-infection with both pathogens. 'CA 336.14.3.0' and 'ICC 14216K' chickpea plants were challenged with Foc race 5 and Ma, either in single or in combined inoculations, and the root proteomes were analyzed by two-dimensional gel electrophoresis using three biological replicates. Pairwise comparisons of treatments indicated that 47 protein spots in 'CA 336.14.3.0' and 31 protein spots in 'ICC 14216K' underwent significant changes in intensity. The responsive protein spots tentatively identified by MALDI TOF-TOF MS (27 spots for 'CA 336.14.3.0' and 15 spots for 'ICC 14216K') indicated that same biological functions were involved in the responses of either chickpea line to Foc race 5 and Ma, although common as well as line-specific responsive proteins were found within the different biological functions. To the best of our knowledge, this is the first study at the root proteome level of chickpea response to a biotic stress imposed by single and joint infections by two major soil-borne pathogens.     

Identification and validation of reference genes for RT‐qPCR analysis in banana (Musa spp.) under Fusarium wilt resistance induction conditions

Banana (Musa spp.) is severely damaged by Fusarium wilt caused by Fusarium oxysporum f. sp. cubense (Foc). Biocontrol by inducing systemic resistance has been considered as one of the most important strategies to improve plant health. Very few studies have investigated appropriate reference gene selection for RT‐qPCR (quantitative real‐time polymerase chain reaction) analysis suitable for conditions of systemic activated resistance. In this study, we assessed over a time‐course the expression of seven candidate reference genes (EF1, TUB, ACT1, ACT2, L2, RPS2 and RAN) for Cavendish cultivar Brazilian (Musa spp. AAA) and dwarf banana cultivar Guangfen No. 1 (Musa spp. ABB) that were inoculated by Bacillus subtilis strain TR21 and Foc. We choose these plants because they are commonly planted in Southern China. Expression stability of the candidate genes was evaluated using various software packages (GeNorm, NormFinder and BestKeeper). L2 and TUB genes displayed maximum stability in Guangfen No. 1. In Brazilian, ACT1 and TUB were the most stable genes. To further validate the suitability of the reference genes identified in this study, the expression of pathogenesis‐related 1 (PR1) gene under TR21 and Foc strains Foc004/Foc009 treatments was also studied. Identified reference genes in this work that are most suitable for normalizing gene expression data in banana under Fusarium wilt resistance induction conditions will contribute to the understanding of disease resistance mechanisms induced by biocontrol strains in banana.     

香蕉枯萎病菌粗毒素对地衣芽胞杆菌生长和培养液上清蛋白组成的影响

本文研究了香蕉枯萎病菌4号生理小种湛江菌株(Foc 4-zj)产生的粗毒素对地衣芽胞杆菌R21菌株生长及其培养液中蛋白组成变化的影响。实验结果表明, Foc 4-zj的粗毒素能够抑制R21菌株的生长, 缩短其生长周期; 减少培养液上清蛋白含量以及改变蛋白质的种类; 低剂量的粗毒素有利于拮抗蛋白的积累, 而高剂量的粗毒素则相反。     

Races and virulence of Fusarium oxysporum f.sp. cubense on local banana cultivars in Kenya

Virulence of 31 Kenyan isolates of Fusarium oxysporum obtained from bananas showing symptoms of Panama disease was tested against the differential banana cvs Bluggoe, Gros Michel, Dwarf Cavendish, and two other local cvs Muraru and Wang'ae. Seventeen isolates were assigned to either race 1 or race 2 of F. oxysporum f.sp. cubense (FOC). Race 4 was not apparent in this sample of 31 isolates from Kenya as none were pathogenic to cv. Cavendish, and no wilted Cavendish have been observed in field surveys in Kenya. Races could not be assigned to 12 isolates as they were virulent on more than one differential cultivar, and two were apparently not pathogenic. All isolates assigned to races 1 and 2 belonged to the VCG bridging complex 0124/5/8/20, but some other isolates belonging to this VCG complex could not be assigned to race. All five isolates assigned to VCG 01212 could not be assigned to known races. Considerable variability thus exists within FOC isolates within this region. Local cultivars of banana showed differential resistance to the pathogen. The interaction of cultivars and isolates on the level of disease was significant. Overall, cv. Wang'ae was the most susceptible to most of the isolates tested, regardless of their race, and could therefore be used as a reference cultivar in pathogenicity tests of isolates of FOC in the East African region. Of the cultivars tested that are widely grown on smallholder farms in Kenya, Muraru was the least susceptible.     

韭菜对香蕉枯萎病菌生长及香蕉枯萎病发生的抑制作用

结合实验室抑菌试验和大棚人工接菌盆栽试验,研究韭菜对香蕉枯萎病菌4号生理小种(Foc4)的拮抗作用及其对香蕉枯萎病发生的防控效果.结果显示:离体条件下,韭菜粗提取液显著抑制Foc4菌丝的生长,造成菌丝变形、细胞的解体;也能显著抑制孢子的萌发并导致孢子失去活性.大棚盆栽试验中,韭菜处理的巴西香蕉苗枯萎病发病率降低70%,病情指数降低86.9%;韭菜处理的广粉1号粉蕉苗枯萎病的发病率降低76.7%,病情指数降低93.4%.研究表明,韭菜对Foc4有很高拮抗效果,而且对香蕉枯萎病有很高的防控作用.     

基于多基因序列分析对尖孢镰孢菌古巴专化型（香蕉枯萎病菌）生理小种的鉴定

由尖孢镰孢菌古巴专化型Fusarium oxysporum f. sp. cubense, Foc引起的香蕉枯萎病是香蕉生产上的毁灭性病害,自1996年以来已对我国华南地区香蕉生产造成了严重危害。传统上香蕉枯萎病菌生理小种的鉴定主要采用人工接种鉴别寄主尔后测定病菌致病性的方法,但实验周期长,且受季节影响。以来自澳大利亚的香蕉枯萎病菌生理小种1号（BW1）、2号（Race 2）、3号（Race 3）以及亚热带4号（BW4）为对照,对分离自我国华南地区主要香蕉产区（广东、广西、海南、福建等省区）的14株香蕉枯萎病菌的单孢菌株进行致病性测定,并结合热带4号小种（TR4）和亚热带4号小种（ST4）的分子特异检测方法,确定其生理小种类型;同时,利用ITS、TEF-1α、IGS、histone H3、β-tubulin等 5个主要用于镰孢菌系统发育学研究的基因,研究不同地区不同来源的Foc菌株之间的亲缘关系及其与非病原尖孢镰孢菌的关系,并评价这5个基因在香蕉枯萎病菌生理小种鉴定上的应用价值。研究结果表明：（1）来源于我国华南地区的4号小种主要为热带4号小种;（2）TEF-1α、IGS、histone H3等3个基因片段能够将Foc中不同生理小种的菌株划分成不同的系统发育谱系,与致病性测定的结果具有对应关系,也能较好地反映尖孢镰孢菌种内菌株的亲缘关系,可用于香蕉枯萎病菌生理小种鉴定;（3）我国Foc 1号生理小种的遗传多样性高于4号生理小种,Foc 1号生理小种的菌系与来自香蕉果实上的非病原尖孢镰孢菌的亲缘关系比其与Foc 4号生理小种的菌系的亲缘关系更近。     

Production of monoclonal antibodies to Fusarium oxysporum f.sp. cubense race 4

The production of monoclonal antibodies (mab) to Fusarium oxysporum f.sp. cubense (Foc ) race 4 is described. Heat-killed conidia of this fungus were toxic to female Balb/c mice, but this toxic reaction was not found with fractionated hyphal walls. A simple and reproducible enzyme immunoassay using a standard 9 cm polystyrene Petri dish as a solid phase was devised for screening culture supernatant fluids. Sixteen stable hybridoma clones secreting mabs of the IgM class were isolated by fusing splenic lymphocytes from immunized female Balb/c mice with P3-NSl-Ag4-l mouse myeloma cells. Monoclonal antibodies produced by eight of the 16 hybridoma clones were selected and the specificity of the mabs was determined by an indirect immunofluorescence test. Of the eight mabs, only one displayed an exceptionally high degree of specificity to the thick-walled chlamydospores of Foc race 4. This specific reactivity allowed differentiation of Foc race 4 from other races.     

Development of a Real-Time Fluorescence Loop-Mediated Isothermal Amplification Assay for Rapid and Quantitative Detection of Fusarium oxysporum f. sp. cubense Tropical Race 4 In Soil

Fusarium oxysporum f. sp. cubense (Foc), the causal agent of Fusarium wilt (Panama disease), is one of the most devastating diseases of banana (Musa spp.). The Foc tropical race 4 (TR4) is currently known as a major concern in global banana production. No effective resistance is known in Musa to Foc, and no effective measures for controlling Foc once banana plants have been infected in place. Early and accurate detection of Foc TR4 is essential to protect banana industry and guide banana planting. A real-time fluorescence loop-mediated isothermal amplification assay (RealAmp) was developed for the rapid and quantitative detection of Foc TR4 in soil. The detection limit of the RealAmp assay was approximately 0.4 pg/µl plasmid DNA when mixed with extracted soil DNA or 10³ spores/g of artificial infested soil, and no cross-reaction with other relative pathogens were observed. The RealAmp assay for quantifying genomic DNA of TR4 was confirmed by testing both artificially and naturally infested samples. Quantification of the soil-borne pathogen DNA of Foc TR4 in naturally infested samples was no significant difference compared to classic real-time PCR (P>0.05). Additionally, RealAmp assay was visual with an improved closed-tube visual detection system by adding SYBR Green I fluorescent dye to the inside of the lid prior to amplification, which avoided the inhibitory effects of the stain on DNA amplification and makes the assay more convenient in the field and could thus become a simple, rapid and effective technique that has potential as an alternative tool for the detection and monitoring of Foc TR4 in field, which would be a routine DNA-based testing service for the soil-borne pathogen in South China.     

7个香蕉品种对枯萎病的抗性评价及3种评价方法的对比

[目的] 评价香蕉自主选育品种对枯萎病病原菌尖孢镰刀菌古巴专化型（Fusarium oxysporum f. sp. cubense）热带4号小种（tropical race 4, Foc TR4）的抗性。[方法] 以浸根法和2种灌注法将Foc TR4接种于不同抗性的香蕉品种上,分析接种后香蕉的发病情况,比较筛选这3种评价方法,并评价7个香蕉品种的苗期和田间抗性水平。[结果] 采用浓度为2×10⁵ 个·mL^-1的PDB培养基Foc TR4孢子悬浮液灌注法进行香蕉苗期抗性评价更为高效可行;综合苗期和田间抗性评价结果,7个香蕉品种的抗性由高到低为：南天黄>红研3号>红研5号>红研2号>滇蕉1号>巴西蕉>红研1号。[结论] 以南天黄作为高抗品种对照,以巴西蕉作为高感品种对照条件下,红研3号和红研5号抗性为中等抗性偏强,红研2号达到中抗水平,滇蕉1号为感病,红研1号为高度感病。     

香蕉肉桂醇脱氢酶基因的克隆及表达分析

肉桂醇脱氢酶(CAD)在木质素合成过程中起关键作用。通过RACE(rapid-amplification of cDNA ends)方法从香蕉根系cDNA均一化全长文库中获得一个肉桂醇脱氢酶基因,命名为MaCAD1(GenBank登录号为KF582533)。MaCAD1是香蕉MYB基因编码框全长cDNA,包含一个1 077bp的最大开放阅读框(ORF),编码358个氨基酸。蛋白质序列同源比对发现,其含有完整的醇脱氧酶的典型保守结构域,属于典型的CAD蛋白。系统进化树比对分析表明,MaCAD1与水稻OsCAD6(CAD39907)的亲缘关系较近。组织特异性研究表明MaCAD1基因组成型表达于香蕉各个组织。在耐病和感病品种中,MaCAD1均上调表达,但在耐病品种中MaCAD1在所有时间点相对于对照增加的倍数均高于感病品种,表明MaCAD1基因在香蕉的抗病性中起着重要作用,MaCAD1可以作为一个新的响应枯萎病侵染的标记基因。