Petunia floral defensins with unique prodomains as novel candidates for development of fusarium wilt resistance in transgenic banana plants

Antimicrobial peptides are a potent group of defense active molecules that have been utilized in developing resistance against a multitude of plant pathogens. Floral defensins constitute a group of cysteine-rich peptides showing potent growth inhibition of pathogenic filamentous fungi especially Fusarium oxysporum in vitro. Full length genes coding for two Petunia floral defensins, PhDef1 and PhDef2 having unique C-terminal 31 and 27 amino acid long predicted prodomains, were overexpressed in transgenic banana plants using embryogenic cells as explants for Agrobacterium-mediated genetic transformation. High level constitutive expression of these defensins in elite banana cv. Rasthali led to significant resistance against infection of Fusarium oxysporum f. sp. cubense as shown by in vitro and ex vivo bioassay studies. Transgenic banana lines expressing either of the two defensins were clearly less chlorotic and had significantly less infestation and discoloration in the vital corm region of the plant as compared to untransformed controls. Transgenic banana plants expressing high level of full-length PhDef1 and PhDef2 were phenotypically normal and no stunting was observed. In conclusion, our results suggest that high-level constitutive expression of floral defensins having distinctive prodomains is an efficient strategy for development of fungal resistance in economically important fruit crops like banana.     

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.     

Transgenic banana expressing Pflp gene confers enhanced resistance to Xanthomonas wilt disease

Banana Xanthomonas wilt (BXW), caused by Xanthomonas campestris pv. musacearum, is one of the most important diseases of banana (Musa sp.) and currently considered as the biggest threat to banana production in Great Lakes region of East and Central Africa. The pathogen is highly contagious and its spread has endangered the livelihood of millions of farmers who rely on banana for food and income. The development of disease resistant banana cultivars remains a high priority since farmers are reluctant to employ labor-intensive disease control measures and there is no host plant resistance among banana cultivars. In this study, we demonstrate that BXW can be efficiently controlled using transgenic technology. Transgenic bananas expressing the plant ferredoxin-like protein (Pflp) gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of banana. These transgenic lines were characterized by molecular analysis. After challenge with X. campestris pv. musacearum transgenic lines showed high resistance. About 67% of transgenic lines evaluated were completely resistant to BXW. These transgenic lines did not show any disease symptoms after artificial inoculation of in vitro plants under laboratory conditions as well as potted plants in the screen-house, whereas non-transgenic control plants showed severe symptoms resulting in complete wilting. This study confirms that expression of the Pflp gene in banana results in enhanced resistance to BXW. This transgenic technology can provide a timely solution to the BXW pandemic.     

Engineered resistance to tomato spotted wilt virus in transgenic peanut expressing the viral nucleocapsid gene

The nucleocapsid gene of tomato spotted wilt virus Hawaiian L isolate in a sense orientation, and the GUS and NPTII marker genes, were introduced into peanut (Arachis hypogaea cv. New Mexico Valencia A) using Agrobacterium-mediated transformation. Modifications to a previously defined transformation protocol reduced the time required for production of transformed peanut plants. Transgenes were stably integrated into the peanut genome and transmitted to progeny. RNA expression and production of nucleocapsid protein in transgenic peanut were observed. Progeny of transgenic peanut plants expressing the nucleocapsid gene showed a 10- to 15-day delay in symptom development after mechanical inoculations with the donor isolate of tomato spotted wilt virus. All transgenic plants were protected from systemic tomato spotted wilt virus infection. Inoculated non-transformed control plants and plants transformed with a gene cassette not containing the nucleocapsid gene became systemically infected and displayed typical tomato spotted wilt virus symptoms. These results demonstrate that protection against tomato spotted wilt virus can be achieved in transgenic peanut plants by expression of the sense RNA of the tomato spotted wilt virus nucleocapsid gene     

Development of antibiotic marker-free creeping bentgrass resistance against herbicides

Herbicide-resistant creeping bentgrass plants (Agrostis stolonifera L.) without antibiotic-resistant markers were produced by Agrobacterium-mediated transformation. Embryogenic callus tissues were infected with Agrobacterium tumefaciens EHA105, harboring the bar and the CP4-EPSPS genes for bialaphos and glyphosate resistance. Phosphinothricin-resistant calli and plants were selected. Soil-grown plants were obtained at 14-16 weeks after transformation. Genetic transformation of the selected, regenerated plants was validated by PCR. Southern blot analysis revealed that at least one copy of the transgene was integrated into the genome of the transgenic plants. Transgene expression was confirmed by Northern blot. CP4-EPSPS protein was detected by ELISA. Transgenic plants remained green and healthy when sprayed with Basta, containing 0.5% glufosinate ammonium or glyphosate. The optimized Agrobacterium-mediated transformation method resulted in an average of 9.4% transgenic plants. The results of the present study suggest that the optimized marker-free technique could be used as an effective and reliable method for routine transformation, which may facilitate the development of varieties of new antibiotic-free grass species.     

几丁质酶基因导入西瓜植株及其抗病性鉴定研究

利用西瓜有性生殖过程 ,将携带有外源几丁质酶基因的质粒DNA涂抹在授粉后的柱头上 ,使其沿花粉管通道进入到生殖细胞 ,得到转化处理种子。转化T1代植株经除草剂Basta筛选和PCR扩增获得转化植株。对T3 代株系进行田间自然发病和人工接种鉴定获得 3个抗枯萎病的株系。结果表明 :几丁质酶对镰刀菌引起的西瓜枯萎病有一定的抑制作用 ,利用花粉管通道法直接导入西瓜活体植株的技术是可行的。     

Expression of sweet pepper Hrap gene in banana enhances resistance to Xanthomonas campestris pv. musacearum

Banana Xanthomonas wilt (BXW), caused by the bacterium Xanthomonas campestris pv. musacearum, is the most devastating disease of banana in the Great Lakes region of Africa. The pathogen's rapid spread has threatened the livelihood of millions of Africans who rely on banana fruit for food security and income. The disease is very destructive, infecting all banana varieties, including both East African Highland bananas and exotic types of banana. In the absence of natural host plant resistance among banana cultivars, the constitutive expression of the hypersensitivity response-assisting protein (Hrap) gene from sweet pepper (Capsicum annuum) was evaluated for its ability to confer resistance to BXW. Transgenic lines expressing the Hrap gene under the regulation of the constitutive CaMV35S promoter were generated using embryogenic cell suspensions of two banana cultivars: 'Sukali Ndiizi' and 'Mpologoma'. These lines were characterized by molecular analysis, and were challenged with Xanthomonas campestris pv. musacearum to analyse the efficacy of the Hrap gene against BXW. The majority of transgenic lines (six of eight) expressing Hrap did not show any symptoms of infection after artificial inoculation of potted plants in the screenhouse, whereas control nontransgenic plants showed severe symptoms resulting in complete wilting. This study demonstrates that the constitutive expression of the sweet pepper Hrap gene in banana results in enhanced resistance to BXW. We describe the development of transgenic banana varieties resistant to BXW, which will boost the arsenal available to fight this epidemic disease and save livelihoods in the Great Lakes region of East and Central Africa.     

青菜的高效再生和农杆菌介导B.t.及CpTI基因的转化

分别对培养基中适宜的激素组成和AgNO3 添加浓度进行研究 ,建立了青菜下胚轴和子叶外植体的高效再生系统 ,青菜品种“矮抗青”的最高芽分化频率下胚轴可达 6 5 % ,子叶为 5 2 %左右。在此基础上 ,对影响转化频率的不同因素进行了探讨 ,共获 94株卡那霉素抗性植株。对转基因植株总DNA的Southernblot ting分析证明 ,B .t .基因和CpTI基因已整合到青菜植株的细胞核基因组中。经过抗虫性筛选试验 ,从转基因植株的T3 代筛选到 7个转B .t.基因的抗虫纯合株系和 5个转CpTI基因的抗虫纯合株系 ,并进入田间小区青菜抗虫新品种试验     

香蕉植株根区土壤的真菌多样性分析

尖孢镰孢菌古巴专化型(Fusarium oxysporum f.sp.cubense)是香蕉枯萎病的病原菌,该菌是一种土壤习居菌,了解香蕉根区土壤中真菌多样性及镰孢菌属(Fusarium)真菌所占比例,对如何减少土壤中的病原菌、预防香蕉枯萎病的发生有重要的指导意义。该文通过采集不同宿根年限的香蕉健康植株和枯萎病植株的根区土壤,利用高通量测序技术测定土壤样品中的真菌种群。结果表明:(1)同一宿根年限的香蕉植株中,健康植株根区土壤中所获的reads及OTUs数量均高于枯萎病植株,说明健康植株根区土壤的真菌多样性丰富于枯萎病植株。(2)除了一年生香蕉枯萎病植株以担子菌门(Basidiomycota)为主外,其他土壤样品中均以子囊菌门(Ascomycota)为主,其中的丛赤壳科最高相对丰度来自三年生健康植株的根区土壤(26.02%),其次是五年生的枯萎病植株根区土壤(15.56%)。(3)在丛赤壳科中,镰孢菌属在三年生健康植株土壤中的相对丰度最高(2.54%),在其他样品中的相对丰度在0.1%～0.65%之间;在镰孢菌属中,腐皮镰孢菌(Fusarium solani)的相对丰度(0～1.59%之间)高于尖孢镰孢菌(F.oxysporum),尖孢镰孢菌仅占很小的比例(相对丰度0～0.08%之间)。可见,在不同香蕉植株的根区土壤中,健康植株的根区土壤真菌多样性高于枯萎病植株,无论是健康植株还是枯萎病植株的根区土壤中,作为香蕉枯萎病病原菌的镰孢菌属或尖孢镰孢菌的群体均不占主导地位。     

Fusarium oxysporum hijacks COI1-mediated jasmonate signaling to promote disease development in Arabidopsis

Although defense responses mediated by the plant oxylipin jasmonic acid (JA) are often necessary for resistance against pathogens with necrotrophic lifestyles, in this report we demonstrate that jasmonate signaling mediated through COI1 in Arabidopsis thaliana is responsible for susceptibility to wilt disease caused by the root-infecting fungal pathogen Fusarium oxysporum . Despite compromised JA-dependent defense responses, the JA perception mutant coronatine insensitive 1 ( coi1 ), but not JA biosynthesis mutants, exhibited a high level of resistance to wilt disease caused by F. oxysporum . This response was independent from salicylic acid-dependent defenses, as coi1/NahG plants showed similar disease resistance to coi1 plants. Inoculation of reciprocal grafts made between coi1 and wild-type plants revealed that coi1 -mediated resistance occurred primarily through the coi1 rootstock tissues. Furthermore, microscopy and quantification of fungal DNA during infection indicated that coi1 -mediated resistance was not associated with reduced fungal penetration and colonization until a late stage of infection, when leaf necrosis was highly developed in wild-type plants. In contrast to wild-type leaves, coi1 leaves showed no necrosis following the application of F. oxysporum culture filtrate, and showed reduced expression of senescence-associated genes during disease development, suggesting that coi1 resistance is most likely achieved through the inhibition of F. oxysporum -incited lesion development and plant senescence. Together, our results indicate that F. oxysporum hijacks non-defensive aspects of the JA-signaling pathway to cause wilt-disease symptoms that lead to plant death in Arabidopsis.     

天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

天麻抗真菌蛋白(gastrodia antifungal protein简称GAFP)是从我国传统中药天麻(Gastrodia elata B1．)中分离到的一种具有广谱抗真菌活性的蛋白质,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用,因此,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法,将GAFP的基因．gafp转入3个新疆彩色棉品种中,通过田间抗病筛选和分子检测,得到了高抗黄萎病的转基因植株,两株Southem杂交阳性植株LB-5-8和ZB-1—49对黄萎病表现整株免疫。RT-PCR的结果显示,LB-5-8和ZB-1—49中均有gafp的正确转录;离体的抑菌实验也表明,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制,表明了gafp在转基因植株中的正确表达,翻译的产物具有活性。经过进一步选育和扩繁,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。     

天麻抗真菌蛋白基因(gafp)转化彩色棉的研究

天麻抗真菌蛋白（gastrodia antifungal protein简称GAFP）是从我国传统中药天麻（Gastrodia elata Bl.）中分离到的一种具有广谱抗真菌活性的蛋白质,它对许多植物真菌病包括棉花枯萎病、黄萎病等的致病菌离体具有很强的抑制作用,因此,在植物抗真菌病基因工程上有很重要的应用价值。本研究通过花粉管通道法,将GAFP的基因gafp转入3个新疆彩色棉品种中,通过田间抗病筛选和分子检测,得到了高抗黄萎病的转基因植株,两株Southern杂交阳性植株LB-5-8和ZB-1-49对黄萎病表现整株免疫。RT-PCR的结果显示,LB-5-8和ZB-1-49中均有gafp的正确转录;离体的抑菌实验也表明,它们的蛋白粗提物对棉花黄萎病致病菌离体有明显的抑制,表明了gafp在转基因植株中的正确表达,翻译的产物具有活性。经过进一步选育和扩繁,发现转基因彩色棉后代具有稳定的、较强的抗黄萎病能力,本研究为通过植物抗病基因工程的方法防治棉花黄萎病提供了一条新的途径。     

Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection

An Agrobacterium-mediated transformation system with glyphosate selection has been developed for the large-scale production of transgenic plants. The system uses 4-day precultured immature embryos as explants. A total of 30 vectors containing the 5-enol-pyruvylshikimate-3-phosphate synthase gene from Agrobacterium strain CP4 (aroA:CP4), which confers resistance to glyphosate, were introduced into wheat using this system. The aroA:CP4 gene served two roles in this study-selectable marker and gene of interest. More than 3,000 transgenic events were produced with an average transformation efficiency of 4.4%. The entire process from isolation of immature embryos to production of transgenic plantlets was 50-80 days. Transgenic events were evaluated over several generations based on genetic, agronomic and molecular criteria. Forty-six percent of the transgenic events fit a 3:1 segregation ratio. Molecular analysis confirmed that four of six lead transgenic events selected from Agrobacterium transformation contained a single insert and a single copy of the transgene. Stable expression of theAROA:CP4 gene was confirmed by ELISA through nine generations. A comparison of Agrobacterium-mediated transformation to a particle bombardment system demonstrated that the Agrobacterium system is reproducible, has a higher transformation efficiency with glyphosate selection and produces higher quality transgenic events in wheat. One of the lead events from this study, no. 33391, has been identified as a Roundup Ready wheat commercial candidate.     

Arabidopsis defense response against Fusarium oxysporum

The plant fungal pathogen Fusarium oxysporum (Fox) is the causal agent of root rot or wilt diseases in several plant species, including crops such as tomato (Solanum lycopersicum), banana (Musa sapientum) and asparagus (Asparagus officinalis). Colonization of plants by Fox leads to the necrosis of the infected tissues, a subsequent collapse of vascular vessels and decay of the plant. Plant resistance to Fox appears to be monogenic or oligogenic depending on the host. Perception of Fox by plants follows the concept of elicitor-induced immune response, which in turn activates several plant defense signaling pathways. Here, we review the Fox-derived elicitors identified so far and the interaction among the different signaling pathways mediating plant resistance to Fox.     

海南省香蕉枯萎病病原菌的鉴定

香蕉枯萎病在海南省为首次报道。在Komada改良培养基鉴定的基础上,用温室人工接种法对采自海南省各市县香蕉种植区的18个香蕉和粉蕉假茎分离物进行鉴定。结果表明香蕉枯萎病菌的两种分离物在培养特性和致病性上存在明显区别,分离自粉蕉的12个菌株为1号生理小种,而分离自香蕉的6个菌株为4号生理小种。     

兔防御素NP-1基因在转基因番茄中表达的初步研究

兔防御素ＮＰ－１是α－防御素的一种,含３３个氨基酸残基。最初从兔子的多形核嗜中性细胞中分离出来。它对革兰氏阴性菌、革兰氏阳性菌、分枝杆菌、真菌、被膜病毒以及ＨＩＶ病毒都有不同程度的抑制作用。兔防御素ＮＰ－１所带阳离子较多,可抗不具代谢活性的靶细胞。实验中将兔防御素ＮＰ－１基因构建到植物表达载体中,通过根瘤农杆菌介导转入番茄,得到了转基因番茄植株。对转基因番茄植株进行了ＰＣＲ、Ｓｏｕｔｈｅｍ杂交、Ｎ