A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco

A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin-induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm-responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre-treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm-induced activation of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain-of-function and loss-of-function studies on MAPK cascade members reveal that the expression of Spm-induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.     

Suppression of defense response in plants by the avrBs3/pthA gene family of Xanthomonas spp

Effector genes of some plant-pathogenic bacteria, including some members of the avrBs3/pthA effector gene family from Xanthomonas spp., confer not only genotype-specific disease resistance but also pathogen aggressiveness or virulence. In addition, some effector gene products suppress induction of a nonspecific (or general) hypersensitive response (HR). To determine whether the Xanthomonas avrBs3/pthA gene family members apl1, avrXa7, or avrXa10 also confer suppressor activity, we introduced constructs with each effector gene into Pseudomonas fluorescens 55 that expressed the entire hrp cluster from P. syringae pv. syringae in cosmid pHIR11. When inoculated to tobacco 'Bright Yellow', P fluorescens (pHIR11) induces the HR and expression of four tobacco defense response genes: HIN1, RbohB, PAL, and PR1. When P. fluorescens double transformants that contained pHIR11 and constructs with apl1, avrXa7, or avrXa10 were infiltrated into tobacco, the HR and expression of three defense response genes, RbohB, PAL, and PR1, were suppressed. The suppression of the HR and defense gene expression was more efficient in the transformants with the apl1 and avrXa7 than the transformant with avrXa10. Although expression of other defense genes was suppressed by the double transformants, HIN1 expression was the same level as was observed after infiltration with P. fluorescens (pHIR11), suggesting that HIN1 may not be involved directly in HR. Taken together, our data suggest that avrXa7, avrXa10, and apl1, when delivered to plant cells by the P. syringae pv. syringae hrp secretion system, can suppress nonhost HR and associated phenotypes.     

Tobacco Rar1, EDS1 and NPR1/NIM1 like genes are required for N-mediated resistance to tobacco mosaic virus

The tobacco N gene confers resistance to tobacco mosaic virus (TMV) and encodes a Toll-interleukin-1 receptor/nucleotide binding site/leucine-rich repeat (TIR-NBS-LRR) class protein. We have developed and used a tobacco rattle virus (TRV) based virus induced gene silencing (VIGS) system to investigate the role of tobacco candidate genes in the N-mediated signalling pathway. To accomplish this we generated transgenic Nicotiana benthamiana containing the tobacco N gene. The transgenic lines exhibit hypersensitive response (HR) to TMV and restrict virus spread to the inoculated site. This demonstrates that the tobacco N gene can confer resistance to TMV in heterologous N. benthamiana. We have used this line to study the role of tobacco Rar1-, EDS1-, and NPR1/NIM1- like genes in N-mediated resistance to TMV using a TRV based VIGS approach. Our VIGS analysis suggests that these genes are required for N function. EDS1-like gene requirement for the N function suggests that EDS1 could be a common component of bacterial, fungal and viral resistance signalling mediated by the TIR-NBS-LRR class of resistance proteins. Requirement of Rar1- like gene for N-mediated resistance to TMV and some powdery mildew resistance genes in barley provide the first example of converging points in the disease resistance signalling pathways mediated by TIR-NBS-LRR and CC-NBS-LRR proteins. The TRV based VIGS approach as described here to study N-mediated resistance signalling will be useful for the analysis of not only disease resistance signalling pathways but also of other signalling pathways in genetically intractable plant systems.     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

拟南芥AtCIPK23基因对烟草抗旱能力的影响

为了探索拟南芥AtCIPK23基因对烟草耐旱能力的影响,对3个转AtCIPK23基因阳性纯合株系KA13、KA14和KA44与野生型烟草K326（对照）进行了自然干旱处理,测定离体叶片的失水速率、叶绿素含量、相对电导率、脯氨酸和可溶性糖含量,并分析了转基因及野生型材料对活性氧的清除能力,对活性氧清除基因NtSOD、NtCAT和NtAPX及干旱胁迫相关基因NtDREB、NtLEA5和NtCDPK2的表达量进行检测。结果表明：（1）转基因烟草离体叶片的失水速率明显低于K326;自然干旱7 d后,野生型K326出现了明显的干旱胁迫症状;干旱7 d进行复水后,转基因株系的复水存活率明显高于K326。（2）转基因株系中的叶绿素、脯氨酸及可溶性糖含量比K326显著提高,电导率则明显降低。（3）野生型烟草K326中H₂O₂的积累量明显高于3个转基因株系,转基因株系中ROS清除机制的3个关键基因NtSOD、NtCAT和NtAPX被诱导上调表达。（4）抗旱相关基因NtDREB、NtLEA5和NtCDPK2仅在转基因烟草中受干旱诱导。研究认为,AtCIPK23基因可能具有提高植物抗旱能力的功能。     

Elicitin-responsive lectin-like receptor kinase genes in BY-2 cells.

The inhibition of elicitor-induced plant defense responses by the protein kinase inhibitors K252a and staurosporine indicates that defense responses require protein phosphorylation. We isolated a cDNA clone encoding Nicotiana tabacum lectin-like receptor protein kinase 1 (NtlecRK1), an elicitor-responsive gene; in tobacco bright yellow (BY-2) cells by a differential display method. NtlecRK forms a gene family with at least three members in tobacco. All three NtlecRK genes potentially encode the N-terminal legume lectin domain, transmembrane domain and C-terminal Ser/Thr-type protein kinase domain. Green fluorescent protein (GFP) fusion showed that the NtlecRK1 protein was located on the plasma membrane. In addition, NtlecRK1 and 3 were responsive to INF1 elicitin and the bacterial elicitor harpin. These results indicate that NtlecRKs are membrane-located protein kinases that are induced during defense responses in BY-2 cells.     

转GhB301基因烟草的防御酶活性及抗病相关基因表达分析

为了明确棉花ERF-B3亚族转录因子基因GhB301在烟草异位表达后(抗枯萎病中)的功能,该研究以过表达GhB301基因烟草和野生型烟草为材料,采用枯萎病菌孢子悬浮液接菌方法,分析病原菌侵染前后防御酶活性变化以及防卫相关基因的表达变化与抗病性的关系。结果显示:(1)棉花枯萎病菌处理15d后,2个转基因株系烟草叶片黄化程度与野生型相比较轻。(2)棉花枯萎病菌处理后,过表达GhB301转基因烟草和野生型烟草叶片过氧化物酶(POD)、苯丙氨酸解氨酶(PAL)、多酚氧化酶(PPO)的活性较未接菌对照显著提高,并且酶活峰值出现均早于野生型材料;转基因材料叶片的POD、PAL、PPO活性均在处理3d后达到峰值,而野生型材料叶片的POD、PAL活性在处理5d后才达到峰值。(3)接种棉花枯萎病菌后活性氧相关基因、乙烯(ET)/茉莉酸(JA)途径相关基因、病程相关基因的表达量在转基因株系OE1和OE2中均受到明显影响。研究推测,GhB301在烟草中的异位表达激活了防卫相关基因的表达,提高了防御酶的活性,从而增强了烟草对枯萎病菌的抗性。     

Markers for hypersensitive response and senescence show distinct patterns of expression

Controlled cellular suicide is an important process that can be observed in various organs during plant development. From the generation of proper sexual organs in monoecious plants to the hypersensitive response (HR) that occurs during incompatible pathogen interactions, programmed cell death (PCD) can be readily observed. Although several biochemical and morphological parameters have been described for various types of cell death in plants, the relationships existing between those different types of PCD events remain unclear. In this work, we set out to examine if two early molecular markers of HR cell death (HIN1 and HSR203J) as well as a senescence marker (SAG12) are coordinately induced during these processes. Our result indicates that although there is evidence of some cross-talk between both cell death pathways, spatial and temporal characteristics of activation for these markers during hypersensitive response and senescence are distinct. These observations indicate that these markers are relatively specific for different cell death programs. Interestingly, they also revealed that a senescence-like process seems to be triggered at the periphery of the HR necrotic lesion. This suggests that cells committed to die during the HR might release a signal able to induce senescence in the neighboring cells. This phenomenon could correspond to the establishment of a second barrier against pathogens. Lastly, we used those cell death markers to better characterize cell death induced by copper and we showed that this abiotic induced cell death presents similarities with HR cell death.     

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.     

Induction of hypersensitive cell death by hydrogen peroxide produced through polyamine degradation in tobacco plants

Screening immediate-early responding genes during the hypersensitive response (HR) against tobacco mosaic virus infection in tobacco (Nicotiana tabacum) plants, we identified a gene encoding ornithine decarboxylase. Subsequent analyses showed that other genes involved in polyamine biosynthesis were also up-regulated, resulting in the accumulation of polyamines in apoplasts of tobacco mosaic virus-infected leaves. Inhibitors of polyamine biosynthesis, alpha-difluoromethyl-ornithine, however, suppressed accumulation of polyamines, and the rate of HR was reduced. In contrast, polyamine infiltration into a healthy leaf induced the generation of hydrogen peroxide and simultaneously caused HR-like cell death. Polyamine oxidase activity in the apoplast increased up to 3-fold that of the basal level during the HR, and its suppression with a specific inhibitor, guazatine, resulted in reduced HR. Because it is established that hydrogen peroxide is one of the degradation products of polyamines, these results indicate that one of the biochemical events in the HR is production of polyamines, whose degradation induces hydrogen peroxide, eventually resulting in hypersensitive cell death.