Avr／Cf互作诱发番茄Pto互作因子Pti编码基因的表达

植物抗病基因Pto和Cf产物具完全不同的结构域,其细胞定位也不同。二决定的抗病性产生机制的异同令人关注。采用两种过敏性反应（hypersensitive response,HR）产生系统研究了Pro互作蛋白Pti4,Pti5和Pti6编码基因在Avr/Cf互作中的时序表达：（1）通过杂交方法获取同含互补基因对Avr4/Cf-4和Avr9/Cf-9的番茄（Lycoper-sicon esculentum Mill.）种子,常温下这些种子发芽后形成的苗产生HR坏死斑。（2）先将Avr/Cf苗置于33℃下培养,此时番茄苗生长正常,不形成HR坏死斑。然后将温度降至25℃,数小时内这些苗即形成HR坏死斑。不同方法研究结果均表明,随Avr/Cf苗中HR坏死斑的形成,Pti4、Pti5和Pti6均受显诱导表达。但它们的表达水平和动态不同,这些结果表明,这些Pti在功能上互补,可能同时涉及Pto和Cf决定性抗性的调节作用。     

Avr/Cf互作诱发番茄Pto互作因子Pti编码基因的表达(英)

植物抗病基因Pto和Cf产物具完全不同的结构域 ,其细胞定位也不同。二者决定的抗病性产生机制的异同令人关注。采用两种过敏性反应 (hypersensitiveresponse ,HR)产生系统研究了Pto互作蛋白Pti4、Pti5和Pti6编码基因在Avr/Cf互作中的时序表达 :(1)通过杂交方法获取同含互补基因对Avr4 /Cf_4和Avr9/Cf_9的番茄 (Lycoper siconesculentumMill.)种子。常温下这些种子发芽后形成的苗产生HR坏死斑。 (2 )先将Avr/Cf苗置于 33℃下培养 ,此时番茄苗生长正常 ,不形成HR坏死斑。然后将温度降至 2 5℃ ,数小时内这些苗即形成HR坏死斑。不同方法研究结果均表明 ,随Avr/Cf苗中HR坏死斑的形成 ,Pti4、Pti5和Pti6均受显著诱导表达。但它们的表达水平和动态不同。这些结果表明 ,这些Pti在功能上互补 ,可能同时涉及Pto和Cf决定性抗性的调节作用。     

水杨酸和乙烯对依赖于Cf基因的过敏坏死的调控作用

通过农杆菌(Agrobacterium tumefaciens)介导的方法将互补Aνr/Cf基因对同时在烟草叶片中表达,可以导致过敏性坏死反应。以水杨酸积累缸失型nahC和乙烯不应型etr1-1转基因烟草植株为材料,对水杨酸和乙烯在依赖于番茄Cf-4和Cf—9基因的过敏坏死中的调控作用进行了比较研究。结果表明,nahG植株产生的依赖于Cf-4的过敏坏死反应强度与野生型相似,依赖于Cf—9的坏死反应则明显轻于野生型。转etr1—1基因植株产生的依赖于Cf-4和Cf—9的坏死反应均轻于野生型,与依赖于Cf-4的坏死反应相比,转基因植株中依赖于Cf—9的坏死反应比野生型的减轻程度更显著。这些结果说明水杨酸可能对依赖于Cf—9的过敏坏死起重要调控作用,但对依赖于Cf-4的无此作用;而乙烯则对两者依赖性过敏坏死均起调控作用。     

TMV-番茄互作中的系统性PCD反应

以往研究证实超敏反应(HR)中的细胞程序化死亡(PCD)是植物对病毒的有效防御手段. 但目前对系统获得性抗性反应(SAR)中的PCD至今还知之甚少. 通过烟草花叶病毒(tobacco mosaic virus, TMV)-番茄(Lycopersicon esculentum)互作系统, 发现TMV的局部侵染能够诱导番茄非接种部位的细胞死亡, 但这些部位的酶联免疫吸附实验(ELISA)中未发现有病毒; 且发生死亡的细胞都表现以下的生物学及分子学特征：染色质浓缩, 形成团块, 并于细胞核边缘分布; 细胞核的TUNEL检测呈阳性; DNA ladder检测出现梯状条带; 线粒体和叶绿体结构破毁; 液泡膜和质膜发生收缩和降解等. 结果说明 TMV对番茄叶片的局部性侵染, 在病毒不发生扩散的情况下, 能引起根尖、茎尖和非接种叶片中的系统性PCD反应, 上述组织中的这种反应在发生时间与同步性上不同, 且在诱发原因和表现形式上与HR-PCD和SAR存在差异.     

不同作物两苗同穴互作育苗的生理生态效应

为了探明不同作物两苗同穴互作育苗提高目的作物幼苗素质的机理,本试验在塑料温棚20-30℃、自然光照条件下,采用532mm×280mm具有200方形孔的塑料育苗盘,用土壤作基质,分别以小麦、玉米、谷子、高粱和目的作物棉花、油菜、番茄、花生、牡丹、烟草同穴播种,研究了互作育苗对育苗土壤微生物、酶活性及根系分泌物的影响,以及对目的作物幼苗根系活力、叶片可溶性糖含量和ATP含量的影响,结果表明:随互作苗的加入,育苗土壤中细菌数量显著增加52.80％-102.76％、放线菌数量显著增加34.11％-76.48％、真菌数量显著降低44.33％-56.14％;所测土壤酶活性显著提高,其中脱氢酶活性显著提高30.57％-66.37％、中性磷酸酶活性显著提高38.17％-54.37％、转化酶活性显著提高23.74％-35.04％、脲酶酶活性显著提高60.25％-85.47％;所测根系分泌物积累量显著减少,其中2,4-二叔丁基苯酚显著减少32.80％-51.65％、2,6-二叔丁基苯酚显著减少36.60％-56.59％、邻苯二甲酸二丁酯显著减少10.42％-49.99％、9-16碳烯酸甲酯显著减少25.62％-55.59％;目的作物则表现为根系活力、叶片可溶性糖含量和ATP含量显著提高,增加了目的作物幼苗根重、苗重和侧根数,离床存活期延长,栽后缓苗期缩短,表现互作促进.在所有互作处理中,以棉花+小麦、棉花+谷子、油菜+谷子、番茄+小麦、番茄+谷子、花生+小麦、花生+谷子、牡丹+谷子、烟草+谷子处理中目的作物幼苗素质表现较好.不同作物两苗同穴互作育苗改善了育苗土壤微生物数量和结构,这可能是提高土壤酶活性和降低土壤有害根系分泌物积累的主要原因,进而提高了目的作物幼苗素质.     

番茄花叶病毒移动蛋白和番茄抗病基因Tm-2^2的互作诱导烟草转化体程序性细胞死亡

番茄的抗病基因Tm -2 2 与番茄花叶病毒 (ToMV)的移动蛋白MP基因是一对互作的基因 ,Tm- 2 2 基因和ToMV MP基因同时在烟草中表达 ,并分别获得单一基因整合的纯合转化体植株。病毒接种试验表明 ,Tm -2 2 基因转化体与Tm- 2 2 番茄对Tobamavirus病毒的特异抗性结果一致 ;Tm -2 2 转基因植株和ToMV MP转基因植株杂交试验及其农杆菌注射试验均证明 :(1)Tm -2 2 基因与ToMV- MP在转基因烟草上保持“基因对基因”的互作关系 ;(2 )在外源乙烯的参与下 ,ToMV的移动蛋白与Tm -2 2 基因编码蛋白的互作能够诱导转化体程序性细胞死亡。这一结果为今后研究Tm -2 2 与MP互作的分子机制奠定了基础。     

番茄花叶病毒移动蛋白和番茄抗病基因Tm-22的互作诱导烟草转化体程序性细胞死亡

番茄的抗病基因Tm22与番茄花叶病毒（ToMV）的移动蛋白MP基因是一对互作的基因,Tm22基因和ToMVMP基因同时在烟草中表达, 并分别获得单一基因整合的纯合转化体植株。病毒接种试验表明,Tm22基因转化体与Tm22番茄对Tobamavirus病毒的特异抗性结果一致;Tm22转基因植株和ToMVMP转基因植株杂交试验及其农杆菌注射试验均证明： （1）Tm22基因与ToMVMP在转基因烟草上保持“基因对基因"的互作关系; （2）在外源乙烯的参与下,ToMV的移动蛋白与Tm22基因编码蛋白的互作能够诱导转化体程序性细胞死亡。这一结果为今后研究Tm22与MP互作的分子机制奠定了基础。     

桔梗种子萌发对低温、干旱及互作胁迫的响应

以药用植物桔梗种子为材料,研究了低温、干旱及其互作对种子萌发和幼苗生长的影响,旨在探索在春寒干旱条件下其种子萌发对策及生态适应性,为桔梗野生种群恢复及人工栽培群体构建提供依据.结果表明,温度、干旱及其互作对桔梗种子萌发和幼苗生长均有显著影响.0-10％ PEG水分条件下,在变温10/20℃及恒温15-25℃下桔梗种子能够良好萌发(10％ PEG、20℃除外),而15％ PEG可显著抑制萌发.低温(10℃)下种子萌发始时间延迟,发芽率降低.随干旱胁迫程度的增加,10℃发芽率呈先升后降趋势,而其它温度下,发芽率和发芽指数均呈下降趋势.随温度的降低,幼苗根长、茎长及鲜重均呈下降趋势,且幼根生长对温度变化更为敏感.     

植物病毒载体在植病互作研究中的应用

在植物与病原菌互作的研究中,植物抗性基因和病原菌无毒基因的研究是两个重要的热点。利用植物病毒沉默载体构建的VIGS(Virus Induced Gene Silencing)体系研究植物的防御机制;利用植物病毒表达载体克隆和研究病原菌的无毒基因,将使我们更深刻地理解植物和病原菌互作的分子机理,最终为培育番茄白粉病持久抗性品种打下理论基础。对植物病毒载体的研究进行了综述并就我们承担的课题进行了讨论。     

稻米碾磨品质性状遗传主效应及其与环境互作的遗传分析

采用包括遗传主效应和基因型与环境互作效应的种子三倍体遗传模型,分析了不同环境条件下籼稻稻米碾磨品质的遗传特性。结果表明,各碾磨品质性状除了受制于种子基因效应、细胞质效应和母体植株基因效应等遗传主效应外,还会明显受到各遗传效应与环境互作效应的影响。其中精米重和精米率两个性状的表现主要是以遗传主效应为主,而糙米重和糙米率则以基因型ｘ环境互作效应为主。在各遗传体系中,除了精米率性状的种子直接遗传效应与环境的互作效应略大于母体植株遗传效应与环境的互作效应外,其他碾磨品质性状的母体植株基因的表达受环境条件的影响程度要大于种子基因。细胞质ｘ环境互作效应对精米重和糙米率的影响也较为重要。遗传效应预测值结果表明,Ｐ７和Ｐ８两个亲本的遗传主效应表现突出,可以明显改良杂交后代多数碾磨品质性状。Ｐ１和Ｐ２两个亲本的碾磨品质性状预测值在两年中的环境互作效应较为－致,不易受到外界条件的影响。上述亲本在水稻品质育种中可加以利用。     

An NB-LRR protein required for HR signalling mediated by both extra- and intracellular resistance proteins

Tomato (Solanum lycopersicum) Cf resistance genes confer hypersensitive response (HR)-associated resistance to strains of the pathogenic fungus Cladosporium fulvum that express the matching avirulence (Avr) gene. Previously, we identified an Avr4-responsive tomato (ART) gene that is required for Cf-4/Avr4-induced HR in Nicotiana benthamiana as demonstrated by virus-induced gene silencing (VIGS). The gene encodes a CC-NB-LRR type resistance (R) protein analogue that we have designated NRC1 (NB-LRR protein required for HR-associated cell death 1). Here we describe that knock-down of NRC1 in tomato not only affects the Cf-4/Avr4-induced HR but also compromises Cf-4-mediated resistance to C. fulvum. In addition, VIGS using NRC1 in N. benthamiana revealed that this protein is also required for the HR induced by the R proteins Cf-9, LeEix, Pto, Rx and Mi. Transient expression of NRC1(D481V), which encodes a constitutively active NRC1 mutant protein, triggers an elicitor-independent HR. Subsequently, we transiently expressed this auto-activating protein in N. benthamiana silenced for genes known to be involved in HR signalling, thereby allowing NRC1 to be positioned in an HR signalling pathway. We found that NRC1 requires RAR1 and SGT1 to be functional, whereas it does not require NDR1 and EDS1. As the Cf-4 protein requires EDS1 for its function, we hypothesize that NRC1 functions downstream of EDS1. We also found that NRC1 acts upstream of a MAP kinase pathway. We conclude that Cf-mediated resistance signalling requires a downstream NB-LRR protein that also functions in cell death signalling pathways triggered by other R proteins.     

Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans

The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance.     

The tomato Cf-9 disease resistance gene functions in tobacco and potato to confer responsiveness to the fungal avirulence gene product avr 9

The Cf-9 gene encodes an extracytoplasmic leucine-rich repeat protein that confers resistance in tomato to races of the fungus Cladosporium fulvum that express the corresponding avirulence gene Avr 9. We investigated whether the genomic Cf-9 gene functions in potato and tobacco. Transgenic tobacco and potato plants carrying Cf-9 exhibit a rapid hypersensitive cell death response (HR) to Avr 9 peptide injection. Cf 9 tobacco plants were reciprocally crossed to Avr 9-producing tobacco. A developmentally regulated seedling lethal phenotype occurred in F1 progeny when Cf9 was used as the male parent and Avr 9 as the female parent. However, when Cf9 was inherited in the maternal tissue and a heterozygous Avr 9 plant was used as the pollen donor, a much earlier reaction was caused, leading to no germination of any F1 seed. Detailed analysis of the Avr 9-induced responses in Cf 9 tobacco leaves revealed that (1) most mesophyll cells died within 3 hr (compared with 12 to 16 hr in tomato); (2) the macroscopic HR was visible at an Avr 9 titer five times lower than that which caused visible symptoms in tomato; (3) the HR invariably extended into noninjected panels of the tobacco leaf; (4) no HR occurred in leaves of young tobacco plants; (5) in older plants, the HR was dramatically enhanced by sequential Avr 9 challenges; and (6) coexpression of a salicylate hydroxylase transgene (nahG) from Pseudomonas putida reduced the severity of the macroscopic leaf HR and also restored germination to Cf 9 x 35S:Avr 9 F1 seedlings. Simultaneous introduction of Cf-9 homologs (Hcr 9-9 genes A and B or D) along with the native Cf-9 gene did not alter the responses that were specifically induced by Avr 9. Various ways to use the Cf-9-Avr 9 gene combination to engineer broad-spectrum disease resistance in several solanaceous species are discussed.     

Agroinfiltration is a versatile tool that facilitates comparative analyses of Avr9/Cf-9-induced and Avr4/Cf-4-induced necrosis

The avirulence genes Avr9 and Avr4 from the fungal tomato pathogen Cladosporium fulvum encode extracellular proteins that elicit a hypersensitive response when injected into leaves of tomato plants carrying the matching resistance genes, Cf-9 and Cf-4, respectively. We successfully expressed both Avr9 and Avr4 genes in tobacco with the Agrobacterium tumefaciens transient transformation assay (agroinfiltration). In addition, we expressed the matching resistance genes, Cf-9 and Cf-4, through agroinfiltration. By combining transient Cf gene expression with either transgenic plants expressing one of the gene partners, Potato virus X (PVX)-mediated Avr gene expression, or elicitor injections, we demonstrated that agroinfiltration is a reliable and versatile tool to study Avr/Cf-mediated recognition. Significantly, agroinfiltration can be used to quantify and compare Avr/Cf-induced responses. Comparison of different Avr/Cf-interactions within one tobacco leaf showed that Avr9/Cf-9-induced necrosis developed slower than necrosis induced by Avr4/Cf-4. Quantitative analysis demonstrated that this temporal difference was due to a difference in Avr gene activities. Transient expression of matching Avr/Cf gene pairs in a number of plant families indicated that the signal transduction pathway required for Avr/Cf-induced responses is conserved within solanaceous species. Most non-solanaceous species did not develop specific Avr/Cf-induced responses. However, co-expression of the Avr4/Cf-4 gene pair in lettuce resulted in necrosis, providing the first proof that a resistance (R) gene can function in a different plant family.     

Specific recognition of AVR4 and AVR9 results in distinct patterns of hypersensitive cell death in tomato, but similar patterns of defence-related gene expression

Hypersensitive cell death occurs in tomato seedlings that are derived from a cross between plants that express a resistance (Cf) gene against the pathogenic fungus Cladosporium fulvum and plants that contain the matching avirulence (Avr) gene originating from this fungus. The pattern of Cf-9/Avr9- and Cf-4/Avr4-induced necrosis in these F₁ seedlings was found to differ significantly. Macroscopic observation revealed that in F₁ tomato seedlings containing both Cf-9 and Avr9, numerous necrotic spots developed that were scattered over the entire cotyledon, while the midvein and primary veins remained unaffected. In seedlings containing both Cf-4 and Avr4, however, initially only one or a few necrotic spots developed on each cotyledon, in most cases in the midvein and occasionally in primary veins. Subsequently, these spots turned rapidly into lesions that enlarged along the midvein and primary veins, eventually causing the cotyledons to wilt and abscise. These observations were confirmed by detailed histological studies. Production of the AVR proteins in adult tomato plants carrying the matching Cf gene, employing potato virus X, resulted in similar patterns of necrosis. RNA gel blot analysis demonstrated that both Avr4 and Avr9, controlled by the CaMV 35S promoter, were highly expressed in seedlings already at one day post-emergence, indicating that the distinct necrotic patterns are not due to differences in Avr expression levels. We have analysed the expression of many genes involved in defence signalling pathways and the defence response itself, during the onset of the Cf/Avr-initiated hypersensitive response (HR). Although most of the genes were expressed stronger and faster in Cf-4/Avr4 seedlings than in Cf-9/Avr9 seedlings at the onset of HR, no significant qualitative differences in the expression of genes involved in downstream signalling were observed when Cf-4/Avr4- and Cf-9/Avr9-induced defence responses were compared.     

The F-box protein ACRE189/ACIF1 regulates cell death and defense responses activated during pathogen recognition in tobacco and tomato

Virus-induced gene silencing identified the Avr9/Cf-9 RAPIDLY ELICITED gene ACRE189 as essential for the Cf-9- and Cf-4-mediated hypersensitive response (HR) in Nicotiana benthamiana. We report a role for ACRE189 in disease resistance in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). ACRE189 (herein renamed Avr9/Cf-9-INDUCED F-BOX1 [ACIF1]) encodes an F-box protein with a Leu-rich-repeat domain. ACIF1 is widely conserved and is closely related to F-box proteins regulating plant hormone signaling. Silencing of tobacco ACIF1 suppressed the HR triggered by various elicitors (Avr9, Avr4, AvrPto, Inf1, and the P50 helicase of Tobacco mosaic virus [TMV]). ACIF1 is recruited to SCF complexes (a class of ubiquitin E3 ligases), and the expression of ACIF1 F-box mutants in tobacco compromises the HR similarly to ACIF1 silencing. ACIF1 affects N gene-mediated responses to TMV infection, including lesion formation and salicylic acid accumulation. Loss of ACIF1 function also reduced confluent cell death induced by Pseudomonas syringae pv tabaci. ACIF1 silencing in Cf9 tomato attenuated the Cf-9-dependent HR but not Cf-9 resistance to Cladosporium fulvum. Resistance conferred by the Cf-9 homolog Cf-9B, however, was compromised in ACIF1-silenced tomato. Analysis of public expression profiling data suggests that Arabidopsis thaliana homologs of ACIF1 (VFBs) regulate defense responses via methyl jasmonate- and abscisic acid-responsive genes. Together, these findings support a role of ACIF1/VFBs in plant defense responses.