Programmed cell death suppression in transformed plant tissue by tomato cDNAs identified from an <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-based functional screen

The genetic regulation of programmed cell death (PCD) is well characterized in animal systems, but largely unresolved in plants. This research was designed to identify plant genes that can suppress PCD triggered in plants by Fumonisin B1 (FB1). Agrobacterium rhizogenes was used to transform individual members of a cDNA library into tomato roots, which were then screened for resistance to FB1. Cellular changes elicited during FB1-induced PCD include chromatin condensation, fragmentation into pycnotic DNA bodies, TUNEL positive reactions, ROS accumulation, and eventual loss of membrane integrity. Several cDNA library members collectively overexpressed in a transformed root population revealed PCD suppressive action and were recovered by PCR. One of the FB1 suppressive genes was homologous to metallothionein, and shared sequence homology to the animal ortholog reported to suppress PCD through interference with formation or activity of reactive oxygen species (ROS). The metallothionein recovered in this screen suppressed ROS accumulation in FB1-treated roots and prevented symptoms of PCD. Anti-PCD genes recovered by this screen represent potential sources of resistance to PCD-dependent plant diseases, while the screen should be useful to identify genes capable of suppressing PCD triggered by other effectors, including those expressed by root pathogens during infection.     

A local accumulation of the Ralstonia solanacearum PopA protein in transgenic tobacco renders a compatible plant–pathogen interaction incompatible

Plants activate disease resistance responses when they recognize pathogen-derived molecules (elicitors). Frequently, recognition results in a hypersensitive response (HR), which is characterized by local host cell death at the infection site. Here we describe a genetic engineering approach to generate an HR in plants, whether or not an invading micro-organism produces a recognized elicitor. To that aim we created transgenic tobacco plants in which the pathogen-inducible promoter of the hsr203J gene from tobacco controls the expression of the popA elicitor gene from Ralstonia solanacearum. Because PopA itself also induces the hsr203J promoter, transgenic plants rapidly accumulate the bacterial elicitor in the pathogen infection sites. The elicitor becomes converted in plant tissues into its fully active derivatives PopA1-PopA3, showing that the previously observed processing events are not dependent on the bacterial type III secretion system. The outcome of induced PopA accumulation is a localized HR and a high degree of resistance of the transgenic plants to an oomycete pathogen. The system is functional in hybrids between different tobacco varieties, and we show that the engineered resistance, but not the associated cell death, is dependent on the salicylic acid signalling cascade. Although the approach is powerful in generating oomycete resistance, the induced HR might affect plant health. Its application thus requires a careful selection of individual transgenic lines and trials with various pathogens.     

The leucine-rich repeat (LRR) protein, CaLRR1, interacts with the hypersensitive induced reaction (HIR) protein, CaHIR1, and suppresses cell death induced by the CaHIR1 protein

Leucine-rich repeat proteins (LRRs) function in a number of signal transduction pathways via protein–protein interactions. The gene encoding a small protein of pepper, CaLRR1 , is specifically induced upon pathogen challenge and treatment with pathogen-associated molecular patterns (PAMPs). We identified a pepper hypersensitive induced reaction (CaHIR1) protein that interacts with the LRR domain of the CaLRR1 protein using yeast two-hybrid screening. Ectopic expression of the pepper CaHIR1 gene induces cell death in tobacco and Arabidopsis, indicating that the CaHIR1 protein may be a positive regulator of HR-like cell death. Because transformation is very difficult in pepper plants, we over-expressed CaLRR1 and CaHIR1 in Arabidopsis to determine cellular functions of the two genes. The over-expression of the CaHIR1 gene, but not the CaLRR1 gene, in transgenic Arabidopsis confers disease resistance in response to Pseudomonas syringae infection, accompanied by the strong expression of PR genes, the accumulation of both salicylic acid and H₂O₂, and K⁺ efflux in plant cells. In Arabidopsis and tobacco plants over-expressing both CaHIR1 and CaLRR1 , the CaLRR1 protein suppresses not only CaHIR1 -induced cell death, but also PR gene expression elicited by CaHIR1 via its association with HIR protein. We propose that the CaLRR1 protein functions as a novel negative regulator of CaHIR1-mediated cell death responses in plants.     

A tomato metacaspase gene is upregulated during programmed cell death in<Emphasis Type="Italic"> Botrytis cinerea</Emphasis>-infected leaves

Programmed cell death (PCD) in plant cells is often accompanied by biochemical and morphological hallmarks similar to those of animal apoptosis. However, orthologs of animal caspases, cysteinyl aspartate-specific proteases that constitute the core component of animal apoptosis, have not yet been identified in plants. Recent studies have revealed the presence of a family of genes encoding proteins with distant homology to mammalian caspases, designated metacaspases, in the Arabidopsis thaliana genome. Here, we describe the isolation of LeMCA1, a type-II metacaspase cDNA clone from tomato (Lycopersicon esculentum Mill.). BLAST analysis demonstrated that the LeMCA1 gene is located in close vicinity of several genes that have been linked with PCD. Southern analysis indicated the existence of at least one more metacaspase in the tomato genome. LeMCA1 mRNA levels rapidly increased upon infection of tomato leaves with Botrytis cinerea, a fungal pathogen that induces cell death in several plant species. LeMCA1 was not upregulated during chemical-induced PCD in suspension-cultured tomato cells.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.     

Molecular characterization of rice sphingosine-1-phosphate lyase gene OsSPL1 and functional analysis of its role in disease resistance response

Key message

Our results indicate that overexpression of OsSPL1 in transgenic tobacco plants attenuated disease resistance and facilitated programmed cell death.

Abstract

Long-chain base phosphates including sphingosine-1-phosphate have been shown to act as signaling mediators in regulating programmed cell death (PCD) and stress responses in mammals. In the present study, we characterized a rice gene OsSPL1, encoding a putative sphingosine-1-phosphate lyase that is involved in metabolism of sphingosine-1-phosphate. Expression of OsSPL1 was down-regulated in rice plants after treatments with salicylic acid, benzothiadiazole and 1-amino cyclopropane-1-carboxylic acid, but was induced by infection with a virulent strain of Magnaporthe oryzae, the causal agent of rice blast disease. Transgenic tobacco lines with overexpression of OsSPL1 were generated and analyzed for the possible role of OsSPL1 in disease resistance response and PCD. The OsSPL1-overexpressing tobacco plants displayed increased susceptibility to infection of Pseudomonas syringae pv. tabaci (Pst), the causal agent of wildfire disease, showing severity of disease symptom and bacterial titers in inoculated leaves, and attenuated pathogen-induced expression of PR genes after infection of Pst as compared to the wild-type and vector-transformed plants. Higher level of cell death, as revealed by dead cell staining, leakage of electrolyte and expression of hypersensitive response indicator genes, was observed in the OsSPL1-overexpressing plants after treatment with fumonisin B1, a fungal toxin that induces PCD in plants. Our results suggest that OsSPL1 has different functions in regulating disease resistance response and PCD in plants.     

转基因烟草中Bt毒蛋白基因的表达行为

Bt toxin genes were the insecticidal genes most widely used in genetic engineering of pest resistant plant, were of important significance to study their expression behavior in transgenic plants. In this work, a plant expression vector, pBinMoBc, was constructed. It contained the Cry IA(c) gene under control of chimeric OM promoter and the Ω factor. The vector was transferred into tobacco (Nicotiana tabacum L.) plant via Agrobacterium-mediated transformation. ELISA assay showed that the expression levels of the Cry IA(c) gene in transgenic tobacco plants were significantly higher than that in wild-type tobacco plants. The highest could be up to 0.255% of total soluble proteins; the expression level of CryIA(c) gene in transgenic tobacco plant was changeable during the development stages of tobacco plant. Bioassay showed that pBinMoBc transgenic tobacco plants had more notable insecticidal activity than the wild-type tobacco plants. The above results indicated that pBinMoBc was an effective pest-resistent plant expression vector. This study would be very helpful in screening transgenic cotton with high resistance to cotton bollworm (Heliothis armigeva Hubner).     

OsLSD1, a rice zinc finger protein, regulates programmed cell death and callus differentiation

The Arabidopsis LSD1 and LOL1 proteins both contain three conserved zinc finger domains and have antagonistic effects on plant programmed cell death (PCD). In this study, a rice (Oryza sativa) functional homolog of LSD1, designated OsLSD1, was identified. The expression of OsLSD1 was light-induced or dark-suppressed. Overexpression of OsLSD1 driven by the cauliflower mosaic virus 35S promoter accelerated callus differentiation in transformed rice tissues and increased chlorophyll b content in transgenic rice plants. Antisense transgenic rice plants exhibited lesion mimic phenotype, increased expression of PR-1 mRNA, and an accelerated hypersensitive response when inoculated with avirulent isolates of blast fungus. Both sense and antisense transgenic rice plants conferred significantly enhanced resistance against a virulent isolate of blast fungus. Moreover, ectopic overexpression of OsLSD1 in transgenic tobacco (Nicotiana tabacum) enhanced the tolerance to fumonisins B1 (FB1), a PCD-eliciting toxin. OsLSD1 green fluorescent protein fusion protein was located in the nucleus of tobacco cells. Our results suggest that OsLSD1 plays a negative role in regulating plant PCD, whereas it plays a positive role in callus differentiation.     

Coordinated regulation of genes for secretion in tobacco at late developmental stages: association with resistance against oomycetes

Besides the systemic acquired resistance (SAR) induced in response to microbial stimulation, host plants may also acquire resistance to pathogens in response to endogenous stimuli associated with their own development. In tobacco (Nicotiana tabacum), the vegetative-to-flowering transition comes along with a susceptibility-to-resistance transition to the causal agent of black shank disease, the oomycete Phytophthora parasitica. This resistance affects infection effectiveness and hyphal expansion and is associated with extracellular accumulation of a cytotoxic activity that provokes in vitro cell death of P. parasitica zoospores. As a strategy to determine the extracellular events important for restriction of pathogen growth, we screened the tobacco genome for genes encoding secreted or membrane-bound proteins expressed in leaves of flowering plants. Using a signal sequence trap approach in yeast (Saccharomyces cerevisiae), 298 clones were selected that appear to encode for apoplastic, cell wall, or membrane-bound proteins involved in stress response, in plant defense, or in cell wall modifications. Microarray and northern-blot analyses revealed that, at late developmental stages, leaves were characterized by the coordinate up-regulation of genes involved in SAR and in peroxidative cross-linking of structural proteins to cell wall. This suggests the potential involvement of these genes in extracellular events that govern the expression of developmental resistance. The analysis of the influence of salicylic acid on mRNA accumulation also indicates a more complex network for regulation of gene expression at a later stage of tobacco development than during SAR. Further characterization of these genes will permit the formulation of hypotheses to explain resistance and to establish the connection with development.     

Inducible expression of bacterio-opsin in transgenic tobacco and tomato plants

The development of new strategies to enhance resistance of plants to pathogens is instrumental in preventing agricultural losses. Lesion mimic, the spontaneous formation of lesions resembling hypersensitive response lesions in the absence of a pathogen, is a dramatic phenotype occasionally induced upon expression of certain transgenes in plants. These transgenes simulate the presence of a pathogen and, therefore, activate the plant anti-pathogen defense mechanisms and induce a state of systemic resistance. Lesion mimic genes have been successfully used to enhance the resistance of a number of different plants to pathogen attack. However, constitutive expression of these genes in plants is associated with the spontaneous formation of lesions on leaves and stems, reduced growth, and lower yield. We tested the possibility of using a wound-inducible promoter to control the expression of bacterio-opsin (bO), a transgene that confers a lesion mimic phenotype in tobacco and tomato plants when constitutively expressed. We found that plants with inducible expression of bO did not develop spontaneous lesions. Nevertheless, under controlled laboratory conditions, they were found to be resistant to infection by pathogens. The activation of defense mechanisms by the bO gene was not constitutive, and occurred in response to wounding or pathogen infection. Furthermore, wounding of transgenic tobacco plants resulted in the induction of systemic resistance to pathogen attack within 48 h. Our findings provide a promising initial assessment for the use of wound-inducible promoters as a new strategy to enhance pathogen resistance in transgenic crops by means of lesion mimic genes.     

Thaxtomin A induces programmed cell death in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> suspension-cultured cells

Thaxtomin A is the main phytotoxin produced by Streptomyces scabiei, the causative agent of common scab disease of potato. Pathogenicity of S. scabiei is dependent on the production of thaxtomin A which is required for the development of disease symptoms, such as growth inhibition and cell death. We investigated whether thaxtomin A-induced cell death was similar to the hypersensitive cell death that often occurs in response to specific pathogens or phytotoxins during the so-called hypersensitive response (HR). We demonstrated that thaxtomin A induced in Arabidopsis thaliana suspension-cultured cells a genetically controlled cell death that required active gene expression and de novo protein synthesis, and which involved fragmentation of nuclear DNA, a characteristic hallmark of apoptosis. The thaxtomin A-induced form of programmed cell death (PCD) was not a typical HR, since defence responses generally preceding or associated with the HR, such as rapid medium alkalization, oxidative burst and expression of defence-related genes PR1 and PDF1.2, were not observed in plant cells following addition of thaxtomin A. Thaxtomin A has been shown to inhibit cellulose biosynthesis (Scheible et al. in Plant Cell 15:1781, 2003). We showed that isoxaben, a specific inhibitor of cellulose biosynthesis, also induced in Arabidopsis cell suspensions a PCD similar to that induced by thaxtomin A. These data suggested that rapid changes in the plant cell wall composition and organization can induce PCD in plant cells. We discuss how rapid inhibition of cellulose biosynthesis may trigger this process.