DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

Expression of the Bacterial Type III Effector DspA/E in Saccharomyces cerevisiae Down-regulates the Sphingolipid Biosynthetic Pathway Leading to Growth Arrest

Erwinia amylovora, the bacterium responsible for fire blight, relies on a type III secretion system and a single injected effector, DspA/E, to induce disease in host plants. DspA/E belongs to the widespread AvrE family of type III effectors that suppress plant defense responses and promote bacterial growth following infection. Ectopic expression of DspA/E in plant or in Saccharomyces cerevisiae is toxic, indicating that DspA/E likely targets a cellular process conserved between yeast and plant. To unravel the mode of action of DspA/E, we screened the Euroscarf S. cerevisiae library for mutants resistant to DspA/E-induced growth arrest. The most resistant mutants (Δsur4, Δfen1, Δipt1, Δskn1, Δcsg1, Δcsg2, Δorm1, and Δorm2) were impaired in the sphingolipid biosynthetic pathway. Exogenously supplied sphingolipid precursors such as the long chain bases (LCBs) phytosphingosine and dihydrosphingosine also suppressed the DspA/E-induced yeast growth defect. Expression of DspA/E in yeast down-regulated LCB biosynthesis and induced a rapid decrease in LCB levels, indicating that serine palmitoyltransferase (SPT), the first and rate-limiting enzyme of the sphingolipid biosynthetic pathway, was repressed. SPT down-regulation was mediated by dephosphorylation and activation of Orm proteins that negatively regulate SPT. A Δcdc55 mutation affecting Cdc55-PP2A protein phosphatase activity prevented Orm dephosphorylation and suppressed DspA/E-induced growth arrest.     

DspA/E, a type III effector essential for Erwinia amylovora pathogenicity and growth in planta, induces cell death in host apple and nonhost tobacco plants

Erwinia amylovora is responsible for fire blight, a necrotic disease of apples and pears. E. amylovora relies on a type III secretion system (TTSS) to induce disease on hosts and hypersensitive response (HR) on nonhost plants. The DspA/E protein is essential for E. amylovora pathogenicity and is secreted via the TTSS in vitro. DspA/E belongs to a type III effector family that is conserved in several phytopathogenic bacteria. In E. amylovora, DspA/E has been implicated in the generation of an oxidative stress during disease and the suppression of callose deposition. We investigated the fate of DspA/E in planta. DspA/E delivered artificially to apple or tobacco cells by agroinfection induced necrotic symptoms, indicating that DspA/E was probably injected via the TTSS. We confirmed that DspA/E acts as a major cell-death inducer during disease and HR, because the dspA/E mutant is severely impaired in its ability to induce electrolyte leakage in apple and tobacco leaves. Expression of the defense marker gene PR1 was delayed when dspA/E was transiently expressed in tobacco, suggesting that DspA/E-mediated necrosis may be associated with an alteration of defense responses.     

Apple proteins that interact with DspA/E, a pathogenicity effector of Erwinia amylovora, the fire blight pathogen

The disease-specific (dsp) gene dspA/E of Erwinia amylovora encodes an essential pathogenicity effector of 198 kDa, which is critical to the development of the devastating plant disease fire blight. A yeast two-hybrid assay and in vitro protein pull-down assay demonstrated that DspA/E interacts physically and specifically with four similar putative leucine-rich repeat (LRR) receptor-like serine/threonine kinases (RLK) from apple, an important host of E. amylovora. The genes encoding these four DspA/E-interacting proteins of Malus xdomestica (DIPM1 to 4) are conserved in all genera of hosts of E. amylovora tested. They also are conserved in all cultivars of apple tested that range in susceptibility to fire blight from highly susceptible to highly resistant. The four DIPMs have been characterized, and they are expressed constitutively in host plants. In silico analysis indicated that the DIPMs have similar sequence structure and resemble LRR RLKs from other organisms. Evidence is presented for direct physical interaction between DspA/E and the apple proteins encoded by the four identified clones, which may act as susceptibility factors and be essential to disease development. Knowledge of DIPMs and the interaction with DspA/E thus may facilitate understanding of fire blight development and lead to new approaches to control of disease.     

Effects of antisense repression of an Arabidopsis thaliana pyruvate dehydrogenase kinase cDNA on plant development⋆

Pyruvate dehydrogenase kinase (PDHK), a negative regulator of the mitochondrial pyruvate dehydrogenase (PDH) complex (mtPDC), plays a pivotal role in controlling mtPDC activity, and hence, the TCA cycle and cell respiration. This report describes the cloning of a pyruvate dehydrogenase kinase cDNA (AtPDHK) from Arabidopsis thaliana and focuses on the effects of antisense down-regulation of its expression on plant growth and development. The deduced amino acid sequence of AtPDHK exhibits extensive similarity to other plant and mammalian PDHKs, containing conserved domains typical of two-component histidine protein kinases. The Escherichia coli expressed AtPDHK specifically phosphorylated mammalian PDH E1 in a time-dependent manner. Antisense expression of the AtPDHK cDNA led to marked elevation of mtPDC activity in transgenic plants with increases ranging from 137% to 330% compared to control plants. Immunoblot analyses performed with a monoclonal antibody to the E1 mtPDH component (the subunit phosphorylated by PDHK) indicated that the increased mtPDC activity was not the result of an increase in the level of PDH protein. MtPDC from transgenic plants showed a reduced sensitivity to ATP-dependent inactivation compared to that observed in wild-type plants. Collectively, these data suggest that the antisense partial silencing of the negative regulator, PDHK, was responsible for the increased mtPDC activity observed in the antisense PDHK plants. Transgenic plants with partially repressed AtPDHK also displayed altered vegetative growth with reduced accumulation of vegetative tissues, early flower development and shorter generation time. The potential role for AtPDHK gene manipulation in crop improvement is discussed.     

Morphological and physiological characteristics of transgenic tobacco plants expressing expansin genes: <Emphasis Type="Italic">AtEXP10</Emphasis> from <Emphasis Type="Italic">Arabidopsis</Emphasis> and <Emphasis Type="Italic">PnEXPA1</Emphasis> from poplar

Expansins are non-enzymatic plant proteins breaking hydrogen bonds between cellulose microfibrils and hemicellulose polymer matrix. Each plant has many expansin genes, whose protein products participate in the regulation of plant growth and development mainly by regulating cell expansion. To analyze the effects of elevated expansin expression on the plant organ sizes, we cloned the AtEXPA10 gene from Arabidopsis thaliana and PnEXPA1 gene from Populus nigra. Transgenic tobacco plants expressing the target genes were obtained. The obtained transgenic tobacco plants were shown to have significantly larger leaves and longer stems compared to control plants. The flowers were quite insignificantly larger, but at the same time transgenic plants had more flowers. The microscopic studies showed that the organs of AtEXPA10-carrying plants were larger mainly due to stimulated cell proliferation, whereas the overexpression of the PnEXPA1 gene activated cell expansion.     

Phytoremediation of the organic Xenobiotic simazine by p450-1a2 transgenic Arabidopsis thaliana plants

The potential use of human P450-transgenic plants for phytoremediation of pesticide contaminated soils was tested in laboratory and greenhouse experiments. The transgenic P450 CYP1A2 gene Arabidopsis thaliana plants metabolize number of herbicides, insecticides and industrial chemicals. The P450 isozymes CYP1A2 expressed in A. thaliana were examined regarding the herbicide simazine (SIM). Transgenic A. thaliana plants expressing CYP1A2 gene showed significant resistance to SIM supplemented either in plant growth medium or sprayed on foliar parts. The results showed that SIM produces harmful effect on both rosette diameter and primary root length of the wild type (WT) plants. In transgenic A. thaliana lines, the rosette diameter and primary root length were not affected by SIM concentrations used in this experiment. The results indicate that CYP1A2 can be used as a selectable marker for plant transformation, allowing efficient selection of transgenic lines in growth medium and/or in soil-grown plants. The transgenic A. thaliana plants exhibited a healthy growth using doses of up to 250 μmol SIM treatments, while the non-transgenic A. thaliana plants were severely damaged with doses above 50 μmol SIM treatments. The transgenic A. thaliana plants can be used as phytoremediator of environmental SIM contaminants.     

Reduced fire blight susceptibility in apple cultivars using a high‐efficiency CRISPR/Cas9‐FLP/FRT‐based gene editing system

The bacterium Erwinia amylovora, the causal agent of fire blight disease in apple, triggers its infection through the DspA/E effector which interacts with the apple susceptibility protein MdDIPM4. In this work, MdDIPM4 knockout has been produced in two Malus × domestica susceptible cultivars using the CRISPR/Cas9 system delivered via Agrobacterium tumefaciens. Fifty‐seven transgenic lines were screened to identify CRISPR/Cas9‐induced mutations. An editing efficiency of 75% was obtained. Seven edited lines with a loss‐of‐function mutation were inoculated with the pathogen. Highly significant reduction in susceptibility was observed compared to control plants. Sequencing of five potential off‐target sites revealed no mutation event. Moreover, our construct contained a heat‐shock inducible FLP/FRT recombination system designed specifically to remove the T‐DNA harbouring the expression cassettes for CRISPR/Cas9, the marker gene and the FLP itself. Six plant lines with reduced susceptibility to the pathogen were heat‐treated and screened by real‐time PCR to quantify the exogenous DNA elimination. The T‐DNA removal was further validated by sequencing in one plant line. To our knowledge, this work demonstrates for the first time the development and application of a CRISPR/Cas9‐FLP/FRT gene editing system for the production of edited apple plants carrying a minimal trace of exogenous DNA.     

AtZFP1, encoding Arabidopsis thaliana C2H2 zinc-finger protein 1, is expressed downstream of photomorphogenic activation

C₂H₂ zinc-finger proteins play important roles in plant development including floral organogenesis, leaf initiation, lateral shoot initiation, gametogenesis and seed development. The gene for one such protein from Arabidopsis, AtZFP1 (Arabidopsis thalianazinc-finger protein 1), is expressed at high levels in the shoot apex, including the apical meristem, developing leaves and the developing vascular system. In light-grown seedlings, AtZFP1 expression is induced about three days after germination, before the expansion of the true leaves. Dark-grown plants, in which photomorphogenesis is repressed, have no detectable AtZFP1 expression in the shoot apex. Under conditions which induce or mimic photomorphogenic development including growth in the light, shifting dark-grown plants to continuous light or growth on cytokinin in the dark, high levels of AtZFP1 expression are detected. Furthermore, AtZFP1 expression does not depend on active photosynthesis as shown by analysis of plants grown on the carotenoid biosynthetic inhibitor norflurazon. These results are discussed in relation to a possible role for AtZFP1 in shoot development, downstream of photomorphogenic activation.     

A novel chimeric MOMP antigen expressed in Escherichia coli, Arabidopsis thaliana, and Daucus carota as a potential Chlamydia trachomatis vaccine candidate

The major outer membrane protein (MOMP) of Chlamydia trachomatis is a highly antigenic and hydrophobic transmembrane protein. Our attempts to express the full-length protein in a soluble form in Escherichia coli and in transgenic plants failed. A chimeric gene construct of C. trachomatis serovar E MOMP was designed in order to increase solubility of the MOMP protein but with retained antigenicity. The designed construct was successfully expressed in E. coli, in Arabidopsis thaliana, and in Daucus carota. The chimeric MOMP expressed in and purified from E. coli was used as antigen for production of antibodies in rabbits. The anti-chimeric MOMP antibodies recognized the corresponding protein in both E. coli and in transgenic plants, as well as in inactivated C. trachomatis elementary bodies. Transgenic Arabidopsis and carrots were characterized for the number of MOMP chimeric genetic inserts and for protein expression. Stable integration of the transgene and the corresponding protein expression were demonstrated in Arabidopsis plants over at least six generations. Transgenic carrots showed a high level of expression of the chimeric MOMP – up to 3% of TSP.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.     

Small‐molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora

The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)‐inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein‐encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small‐molecule inhibitors that disable T3SS function could be explored to control fire blight disease.