拟南芥NPR1基因的克隆与表达载体的构建

NPR1基因为植物抗病基因表达和系统获得性抗性中的一个关键基因。该文以DNA PCR扩增的方法,从拟南芥基因组DNA中克隆出NPR1基因,通过序列分析,所克隆的 NPR1 基因与报道的基因序列完全一致。将其构建成植物表达载体,为今后植物抗病基因工程的开展奠定了基础。     

Overexpression of Arabidopsis MAP kinase kinase 7 leads to activation of plant basal and systemic acquired resistance

There is a growing body of evidence indicating that mitogen-activated protein kinase (MAPK) cascades are involved in plant defense responses. Analysis of the completed Arabidopsis thaliana genome sequence has revealed the existence of 20 MAPKs, 10 MAPKKs and 60 MAPKKKs, implying a high level of complexity in MAPK signaling pathways, and making the assignment of gene functions difficult. The MAP kinase kinase 7 (MKK7) gene of Arabidopsis has previously been shown to negatively regulate polar auxin transport. Here we provide evidence that MKK7 positively regulates plant basal and systemic acquired resistance (SAR). The activation-tagged bud1 mutant, in which the expression of MKK7 is increased, accumulates elevated levels of salicylic acid (SA), exhibits constitutive pathogenesis-related (PR) gene expression, and displays enhanced resistance to both Pseudomonas syringae pv. maculicola (Psm) ES4326 and Hyaloperonospora parasitica Noco2. Both PR gene expression and disease resistance of the bud1 plants depend on SA, and partially depend on NPR1. We demonstrate that the constitutive defense response in bud1 plants is a result of the increased expression of MKK7, and requires the kinase activity of the MKK7 protein. We found that expression of the MKK7 gene in wild-type plants is induced by pathogen infection. Reducing mRNA levels of MKK7 by antisense RNA expression not only compromises basal resistance, but also blocks the induction of SAR. Intriguingly, ectopic expression of MKK7 in local tissues induces PR gene expression and resistance to Psm ES4326 in systemic tissues, indicating that activation of MKK7 is sufficient for generating the mobile signal of SAR.     

Runaway cell death, but not basal disease resistance, in lsd1 is SA- and NIM1/NPR1-dependent

LSD1 was defined as a negative regulator of plant cell death and basal disease resistance based on its null mutant phenotypes. We addressed the relationship between lsd1-mediated runaway cell death and signaling components required for systemic acquired resistance (SAR), namely salicylic acid (SA) accumulation and NIM1/NPR1. We present two important findings. First, SA accumulation and NIM1/NPR1 are required for lsd1-mediated runaway cell death following pathogen infection or application of chemicals that mimic SA action. This implies that lsd1-dependent cell death occurs 'downstream' of the accumulation of SA. As SA application triggers runaway cell death in lsd1 but not wild-type plants, we infer that LSD1 negatively regulates an SA-dependent signal leading to cell death. Thus SA is both a trigger and a required mediator of lsd1 runaway cell death. Second, neither SA accumulation nor NIM1/NPR1 function is required for the basal resistance operating in lsd1. Therefore LSD1 negatively regulates a basal defense pathway that can act upstream or independently of both NIM1/NPR1 function and SA accumulation following avirulent or virulent pathogen challenge. Our data, together with results from other studies, point to the existence of an SA-dependent 'signal potentiation loop' controlling HR. Continued escalation of signaling in the absence of LSD1 leads to runaway cell death. We propose that LSD1 is a key negative regulator of this signal potentiation.     

Extracellular pyridine nucleotides induce PR gene expression and disease resistance in Arabidopsis

Although it is well known that the pyridine nucleotides NAD and NADP function inside the cell to regulate intracellular signaling processes, recent evidence from animal studies suggests that NAD(P) also functions in the extracellular compartment (ECC). Extracellular NAD(P) [eNAD(P)] can either directly bind to plasma membrane receptors or be metabolized by ecto-enzymes to produce cyclic ADP-ribose and nicotinic acid adenine dinucleotide phosphate, and/or may ADP-ribosylate cell-surface receptors, resulting in activation of transmembrane signaling. In this study, we report that, in plants, exogenous NAD(P) induces the expression of pathogenesis-related ( PR ) genes and resistance to the bacterial pathogen Pseudomonas syringae pv. maculicola ES4326. Chelation of Ca²⁺ by EGTA significantly inhibits the induction of PR genes by exogenous NAD(P), suggesting that exogenous NAD(P) may induce PR genes through a pathway that involves Ca²⁺ signaling. We show that exogenous application of NAD(P) causes accumulation of the defense signal molecule salicylic acid (SA), and induces both SA/NPR1-dependent and -independent PR gene expression and disease resistance. Furthermore, we demonstrate that NAD(P) leaks into the plant ECC after mechanical wounding and pathogen infection, and that the amount of NAD(P) leaking into the ECC after P. syringae pv. tobacco DC3000/ avrRpt2 infection is sufficient for induction of both PR gene expression and disease resistance. We propose that NAD(P) leakage from cells losing membrane integrity upon environmental stress may function as an elicitor to activate plant defense responses. Our data provide evidence that eNAD(P) functions in plant signaling, and illustrate the potential importance of eNAD(P) in plant innate immunity.     

A stress‐inducible sulphotransferase sulphonates salicylic acid and confers pathogen resistance in Arabidopsis

Sulphonation of small molecules by cytosolic sulphotransferases in mammals is an important process in which endogenous molecules are modified for inactivation/activation of their biological effects. Plants possess large numbers of sulphotransferase genes, but their biological functions are largely unknown. Here, we present a functional analysis of the Arabidopsis sulphotransferase AtSOT12 (At2g03760). AtSOT12 gene expression is strongly induced by salt, and osmotic stress and hormone treatments. The T‐DNA knock‐out mutant sot12 exhibited hypersensitivity to NaCl and ABA in seed germination, and to salicylic acid (SA) in seedling growth. In vitro enzyme activity assay revealed that AtSOT12 sulphonates SA, and endogenous SA levels suggested that sulphonation of SA positively regulates SA production. Upon challenging with the pathogen Pseudomonas syringae, sot12 mutant and AtSOT12 over‐expressing lines accumulate less and more SA, respectively, when compared with wild type. Consistent with the changes in SA levels, the sot12 mutant was more susceptible, while AtSOT12 over‐expressing plants are more resistant to pathogen infection. Moreover, pathogen‐induced PR gene expression in systemic leaves was significantly enhanced in AtSOT12 over‐expressing plants. The role of sulphonation of SA in SA production, mobile signalling and acquired systemic resistance is discussed.     

Altered growth and improved resistance of Arabidopsis against Pseudomonas syringae by overexpression of the basic amino acid transporter AtCAT1

Amino acid transporters in plants are crucial for distributing amino acids between plant organs and cellular compartments. The H⁺‐coupled plasma membrane transporter CAT1 (cationic amino acid transporter 1) facilitates the high‐affinity uptake of basic amino acids. The uptake of lysine (Lys) via the roots was not altered in loss‐of‐function mutants, in accordance with the minor expression of CAT1 in roots, but plants ectopically overexpressing CAT1 incorporated Lys at higher rates. Exogenous Lys inhibited the primary root of Arabidopsis, whereas lateral roots were stimulated. These effects were augmented by the presence or absence of CAT1. Furthermore, the total biomass of soil‐grown plants ectopically overexpressing CAT1 was reduced and the time to flowering was accelerated. These effects were accompanied by only minor changes in the overall amino acid profile. Interestingly, CAT1 belongs to a specific small cluster of nitrogen‐containing metabolite transporter genes that are rapidly up‐regulated upon infection with Pseudomonas syringae and that may participate in the systemic response of plants to pathogen attack. The overexpression of CAT1 indeed enhanced the resistance to the hemibiotrophic bacterial pathogen P. syringae via a constitutively activated salicylic acid (SA) pathway, which is consistent with the developmental defects and the resistance phenotype.     

Induced systemic resistance (ISR) against pathogens in the context of induced plant defences

Induced systemic resistance (ISR) of plants against pathogens is a widespread phenomenon that has been intensively investigated with respect to the underlying signalling pathways as well as to its potential use in plant protection. Elicited by a local infection, plants respond with a salicylic-dependent signalling cascade that leads to the systemic expression of a broad spectrum and long-lasting disease resistance that is efficient against fungi, bacteria and viruses. Changes in cell wall composition, de novo production of pathogenesis-related-proteins such as chitinases and glucanases, and synthesis of phytoalexins are associated with resistance, although further defensive compounds are likely to exist but remain to be identified. In this Botanical Briefing we focus on interactions between ISR and induced resistance against herbivores that is mediated by jasmonic acid as a central signalling molecule. While many studies report cross-resistance, others have found trade-offs, i.e. inhibition of one resistance pathway by the other. Here we propose a framework that explains many of the thus far contradictory results. We regard elicitation separately from signalling and from production, i.e. the synthesis of defensive compounds. Interactions on all three levels can act independently from each other.     

Uncoupling PR gene expression from NPR1 and bacterial resistance: characterization of the dominant Arabidopsis cpr6-1 mutant.

In Arabidopsis, NPR1 mediates the salicylic acid (SA)-induced expression of pathogenesis-related (PR) genes and systemic acquired resistance (SAR). Here, we report the identification of another component, CPR 6, that may function with NPR1 in regulating PR gene expression. The dominant CPR 6-1 mutant expresses the SA/NPR1-regulated PR genes (PR-1, BGL 2, and PR-5) and displays enhanced resistance to Pseudomonas syringae pv maculicola ES4326 and Peronospora parasitica Noco2 in the absence of SAR induction. cpr 6-1-induced PR gene expression is not suppressed in the cpr 6-1 npr1-1 double mutant but is suppressed when SA is removed by salicylate hydroxylase. Thus, constitutive PR gene expression in cpr 6-1 requires SA but not NPR1. In addition, resistance to P. s. maculicola ES4326 is suppressed in the cpr 6-1 npr1-1 double mutant, despite expression of PR-1, BGL 2, and PR-5. Resistance to P. s. maculicola ES4326 must therefore be accomplished through unidentified antibacterial gene products that are regulated through NPR1. These results show that CPR 6 is an important regulator of multiple signal transduction pathways involved in plant defense.     

Screening for resistance against Pseudomonas syringae in rice-FOX Arabidopsis lines identified a putative receptor-like cytoplasmic kinase gene that confers resistance to major bacterial and fungal pathogens in Arabidopsis and rice

Approximately 20,000 of the rice-FOX Arabidopsis transgenic lines, which overexpress 13,000 rice full-length cDNAs at random in Arabidopsis, were screened for bacterial disease resistance by dip inoculation with Pseudomonas syringae pv. tomato DC3000 (Pst DC3000). The identities of the overexpressed genes were determined in 72 lines that showed consistent resistance after three independent screens. Pst DC3000 resistance was verified for 19 genes by characterizing other independent Arabidopsis lines for the same genes in the original rice-FOX hunting population or obtained by reintroducing the genes into ecotype Columbia by floral dip transformation. Thirteen lines of these 72 selections were also resistant to the fungal pathogen Colletotrichum higginsianum. Eight genes that conferred resistance to Pst DC3000 in Arabidopsis have been introduced into rice for overexpression, and transformants were evaluated for resistance to the rice bacterial pathogen, Xanthomonas oryzae pv. oryzae. One of the transgenic rice lines was highly resistant to Xanthomonas oryzae pv. oryzae. Interestingly, this line also showed remarkably high resistance to Magnaporthe grisea, the fungal pathogen causing rice blast, which is the most devastating rice disease in many countries. The causal rice gene, encoding a putative receptor-like cytoplasmic kinase, was therefore designated as BROAD-SPECTRUM RESISTANCE 1. Our results demonstrate the utility of the rice-FOX Arabidopsis lines as a tool for the identification of genes involved in plant defence and suggest the presence of a defence mechanism common between monocots and dicots.     

Arabidopsis nonhost resistance gene PSS1 confers immunity against an oomycete and a fungal pathogen but not a bacterial pathogen that cause diseases in soybean

ABSTRACT: BACKGROUND: Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen Phytophthora sojae, the causal organism of the root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. RESULTS: The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1. Responses of pss1 and pen1-1 to P. sojae invasion were distinct and suggest that PSS1 may act at both pre- and post-haustorial levels, while PEN1 acts at the pre-haustorial level against this soybean pathogen. Therefore, PSS1 encodes a new form of nonhost resistance. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. CONCLUSIONS: The study revealed that Arabidopsis PSS1 is a novel nonhost resistance gene that confers a new form of nonhost resistance against both a hemibiotrophic oomycete pathogen, P. sojae and a necrotrophic fungal pathogen, F. virguliforme that cause diseases in soybean. However, this gene does not play any role in the immunity of Arabidopsis to the bacterial pathogen, P. syringae pv. glycinea, which causes bacterial blight in soybean. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.     

Transgenic tomato plants expressing the <Emphasis Type="Italic">Arabidopsis NPR1</Emphasis> gene display enhanced resistance to a spectrum of fungal and bacterial diseases

Development of effective disease-resistance to a broad-range of pathogens in crops usually requires tremendous resources and effort when traditional breeding approaches are taken. Genetic engineering of disease-resistance in crops has become popular and valuable in terms of cost and efficacy. Due to long-lasting and broad-spectrum of effectiveness against pathogens, employment of systemic acquired resistance (SAR) for the genetic engineering of crop disease-resistance is of particular interest. In this report, we explored the potential of using SAR-related genes for the genetic engineering of enhanced resistance to multiple diseases in tomato. The Arabidopsis NPR1 (nonexpresser of PR genes) gene was introduced into a tomato cultivar, which possesses heat-tolerance and resistance to tomato mosaic virus (ToMV). The transgenic lines expressing NPR1 were normal as regards overall morphology and horticultural traits for at least four generations. Disease screens against eight important tropical diseases revealed that, in addition to the innate ToMV-resistance, the tested transgenic lines conferred significant level of enhanced resistance to bacterial wilt (BW) and Fusarium wilt (FW), and moderate degree of enhanced resistance to gray leaf spot (GLS) and bacterial spot (BS). Transgenic lines that accumulated higher levels of NPR1 proteins exhibited higher levels and a broader spectrum of enhanced resistance to the diseases, and enhanced disease-resistance was stably inherited. The spectrum and degree of these NPR1-transgenic lines are more significant compared to that of transgenic tomatoes reported to date. These transgenic lines may be further explored as future tomato stocks, aiming at building up resistance to a broader spectrum of diseases.     

Expression of tobacco class II catalase gene activates the endogenous homologous gene and is associated with disease resistance in transgenic potato plants

We have previously shown that healthy potato plants respond poorly to salicylic acid (SA) for activating disease resistance against the late blight fungal pathogen Phytophthora infestans. However, SA is essential for the establishment of potato systemic acquired resistance (SAR) against P. infestans after treatment with the fungal elicitor arachidonic acid (AA). To understand the molecular mechanisms through which AA induces SA-dependent SAR in potato, we have recently studied the expression of potato class II catalase (Cat2St) in comparison with its tobacco homologue, Cat2Nt, which has previously been shown to bind SA. In the present study, we show that tobacco Cat2Nt is expressed at high levels and accounts for almost half of total SA-binding activity detected in tobacco leaves. In contrast, potato Cat2St is not expressed in healthy leaves, which is associated with the low SA responsiveness of potato plants for activation of disease resistance mechanisms. Upon treatment with AA, expression of potato Cat2St is induced not only in AA-treated leaves, but also in the upper untreated parts of the plants, concomitant with the establishment of SA -dependent SAR to P. infestans. Moreover, expression of the tobacco Cat2Nt gene in transgenic potato plants leads to constitutive expression of the endogenous potato Cat2St gene and is associated with enhanced resistance to P. infestans. These results collectively indicate that plant SA-binding class II catalases may play an important role in the development of disease resistance, possibly by serving as biological targets of SA.