Cloning of various promoters for foreign gene expression in Erwinia ananas

We constructed a promoter-trap plasmid, pEGFP-V1, to isolate various promoters for foreign gene expression in the leaf-colonizing bacterium Erwinia ananas NR-1. A library was constructed in pEGFP-V1 by introducing genomic DNA fragments upstream of the promoterless EGFP gene to transform E. ananas cells. The library, which consisted of 3500 E. ananas transformants was screened for GFP expression. We found nine strong GFP-expressing clones from the library. Furthermore, we characterized the clones by restriction analysis, sequencing, primer extension analysis, and then quantification of promoter activity. Selected promoters, specifically two (PCF9 and PCF53), gave strong gene expression in E. ananas. Our results indicate that pEGFP-V1 is a useful tool for screening DNA fragments with strong promoter activity in E. ananas.     

一个用于鉴定十字花科黑腐病菌Ⅲ型效应物分泌与转运的报告质粒的构建

 通过免疫检测的方法鉴定十字花科黑腐病菌(Xcc)Ⅲ型效应物的分泌和转运,将cyaA基因克隆至带有3×FLAG的pJXG载体上,构建带有3×FLAG和cyaA基因的报告质粒pJAA,并用已鉴定为Ⅲ型效应物的XC_1553启动子区信号区验证该报告质粒。将pJAA1553三亲导入Xcc 8004^*和8004^*ΔhrcV,然后通过Western blotting免疫检测XC_1553的分泌,结果在8004^*/pJAA1553的胞外蛋白中检测到了XC_1553的分泌,而在8004^*ΔhrcV/pJAA1553的胞外蛋白中没有检测到XC_1553的分泌。通过免疫反应定量检测8004^*/pJAA1553和8004^*ΔhrcV/pJAA1553侵染后植物体内的cAMP含量,结果发现8004^*/pJAA1553侵染后植物体内的cAMP含量比8004^*ΔhrcV/pJAA1553侵染后植物体内的cAMP含量高15倍。结果表明,报告质粒pJAA能够应用于鉴定十字花科黑腐病菌的Ⅲ型效应物。     

拟南芥诱导型启动子rd29A的克隆及其功能鉴定

以改善作物抗旱性为目的,采用PCR方法从拟南芥中克隆了诱导型启动子rd29A,序列分析发现克隆的rd29A启动子与已发表的rd29A启动子序列(D13044)的同源性为99.47%。利用DNA重组技术成功构建了rd29A启动子驱动GUS基因的植物表达载体p BI121-rd29-GUS,并通过农杆菌介导法转化烟草,转基因烟草叶片中GUS酶活性的组织化学检测结果表明,rd29A启动子能驱动目的基因的有效表达。因此,可以在后续的马铃薯抗旱转基因研究中直接应用。     

Cross-resistance relationships between benzimidazole fungicides and diethofencarb in Botrytis cinerea and their genetical basis in Ustilago maydis

After nitrosoguanidine- or UV-mutagenesis, three different benzimidazole-resistant phenotypes were isolated on media containing benomyl or a mixture of carbendazim and diethofencarb from wild-type strains of Botrytis cinerea Pers. ex Fr. and Ustilago maydis (D.C.) Corda. Mutants of B. cinerea with moderate (MBr) or low (LBr) resistance to benzimidazoles and high resistance to diethofencarb (Dr) were isolated from the fungicide-mixture-containing medium in low frequency (7–1 × 10^?8). Only benzimidazole-resistant strains highly sensitive to diethofencarb (HBrDs) were identified on benomyl-containing medium at a frequency of 6.6 × 10^?6. Fitness-determining characteristics such as sporulation, germination and germ-tube elongation, were found to be reduced significantly in the mutants of B. cinerea that were resistant to both benzimidazoles and diethofencarb. However, pathogenicity of a MBrDr mutant strain on cucumber seedlings was equal to that of the wild type and a carbendazim + diethofencarb mixture was found to control grey mould caused by the wild type, but was not effective when the plant cotyledons were infected by the mutant strain. Three benzimidazole-resistant phenotypes (HBrDs, HBrDr, MBrDr) were isolated easily in U. maydis from a benomyl-containing medium. In contrast with B. cinerea, only one-tenth of the benzimidazole-resistant strains were sensitive to diethofencarb. Genetic analysis of benzimidazole resistance in U. maydis showed that the three benzimidazole-resistant phenotypes were due to three allelic mutations in a single gene and one of them was responsible for the negative cross-resistance between benzimidazoles and diethofencarb.     

Analysis of the expression patterns of the Arabidopsis thaliana tubulin-1 and Zea mays ubiquitin-1 promoters in rice plants in association with nematode infection

The temporal and spatial expression of three promoters was investigated in transgenic rice plants using promoter-β-glucuronidase (gusA) reporter gene fusions. The promoters studied were ubiquitin-1 (UBI-1) of Zea mays, Cauliflower Mosaic Virus 35S gene (CaMV35S) and a tubulin gene (TUB-1) of Arabidopsis thaliana. The TUB-1 promoter provided 7.32-fold more GUS activity in roots relative to tillers. This was significantly different from the corresponding value of 2.82-fold for CaMV35S but not from that of 4.55-fold for UBI-1, activity of both promoters was higher in the root tips and zone of elongation than mature roots. This younger root tissue represented a declining proportion of the expanding root system with time. Older tissue expressing GUS under control of the TUB-1 promoter showed a steeper decline in activity with time than occurred with the UBI-1 promoter. Nematode infection did not alter the overall pattern of expression from the two promoters, except that the giant cells induced by Meloidogyne incognita retained TUB-1 promoter activity as roots matured. Pratylenchus zeae invaded older root regions than M. incognita and no changes in promoter activity were detected where it fed. The results suggest the TUB-1 promoter has characteristics that favour its use for delivering anti-feedants, such as cysteine proteinase inhibitors, to M. incognita.     

Plasmodiophora brassicae -induced expression of pyk20, an Arabidopsis thaliana gene with glutamine-rich domain

The expression of the pyk20 gene of Arabidopsis thaliana, which encodes a protein containing a glutamine-rich domain is up-regulated during Plasmodiophora brassicae infection. Transgenic A. thaliana plants harbouring a β-glucuronidase (uid A) reporter gene under the control of the pyk20 gene promoter were grown in soil and infected with P. brassicae resting spores. GUS expression in non-infected plants was found in stipules, apical meristem, leaf vascular tissues, vascular tissue of roots and in the root tips. After infection with P. brassicae, GUS staining was observed in the root hairs during primary infection and in galls in roots and hypocotyls during secondary infection phase. GUS expression during primary infection was also detected in a small number (approx. 0.01%) of zoosporangia. Sections of the GUS-stained galls showed reporter gene expression in infected and non-infected tissues. Northern-blot analysis using a pyk20 cDNA clone as a probe confirmed responsiveness of the pyk20 gene to P. brassicae infection.     

Biological control of rice blast by the epiphytic bacterium <Emphasis Type="Italic">Erwinia ananas</Emphasis> transformed with a chitinolytic enzyme gene from an antagonistic bacterium, <Emphasis Type="Italic">Serratia marcescens</Emphasis> strain B2

The gene chiA, encoding for the endochitinase ChiA, was cloned from Serratia marcescens strain B2, a tomato epiphytic bacterium, and introduced into the epiphytic bacterium Erwinia ananas NR-1, isolated from rice phylloplane. The gene chiA was introduced under the control of two types of promoter into a broad-host-range plasmid vector. The vector contained various fragments with promoter activity isolated from E. ananas chromosomal DNA. The constructed vectors were designated pchiA-V1pcf9 and pchiA-V1pcf53 for their respective promoters. E. ananas NR-1 transformed with either of these vectors produced and secreted ChiA. The antifungal activity of ChiA produced by transformed E. ananas NR-1 was demonstrated in vitro by the inhibition of Pyricularia oryzae germ tube elongation such as bursting of the hyphal tip. Transformed E. ananas NR-1 suppressed the incidence of rice blast caused by P. oryzae under greenhouse conditions; however, the magnitude of the suppressive effect depended on which promoter was used. Both transformants and the nontransformant E. ananas NR-1 survived on rice phylloplane. It is expected that the rice epiphytic bacterium E. ananas NR-1 carrying a chitinolytic enzyme gene is an efficient biological control agent against rice blast.     

Hypersensitive response suppression by type III effectors of plant pathogenic bacteria

The suppressor activity of four representative avirulence (avr) genes from the Pseudomonas syringae group against elicitors of a general hypersensitive response (HR) was examined in tobacco leaves inoculated with double transformants of Pseudomonas fluorescens containing both a cosmid plasmid (pHIR11) carrying the hrp gene cluster and a plasmid carrying each avr gene. The double transformants Pf (pHIR11) containing avrB, avrRps4, or avrPto failed to induce an HR, but that carrying avrRpt2 did induce an HR as Pf (pHIR11 + empty vector) did. Thus, some Avr proteins seem to have suppressor activity against a general HR and should promote aggressiveness of the pathogens.     

Infection and colonization of peanut pods by Aspergillus parasiticus and the expression of the aflatoxin biosynthetic gene,nor-1, in infection hyphae

The aflatoxigenic fungi, Aspergillus flavus and A. parasiticus infect a wide variety of crops, all of which produce oil-rich seed. A histological study of the host–pathogen interaction between peanut,Arachis hyphogea , and A. parasiticus was performed in a system where peanuts remained attached to the plant and were inoculated without wounding. For infection studies, a genetically-tagged strain of A. parasiticus, G5, was engineered to harbor the β-glucuronidase (GUS) reporter gene under control of the nor-1 promoter from the aflatoxin biosynthetic pathway. There was a similar temporal pattern of aflatoxin B1 production and appearance of GUS activity in cultures ofA. parasiticus G5. This strain was used to follow infection and aflatoxin production during colonization of undamaged, drought-stressed peanuts. The fungus colonized all tissues of the peanut pod and appeared to gain ingress through the corky layer of the pericarp. Both intra- and inter-cellular colonization were observed. Fungal colonization of the cotyledons resulted in visible depletion of storage bodies within cells. Two morphologically distinct types of hyphae, wider hyphae and narrower hyphae, were seen throughout the pod tissues. Statistical analysis revealed that the narrower hyphae were significantly more likely to produce GUS activity than wider ones. GUS activity was found in hyphae infecting the pericarp, embryo and cotyledons indicating expression of aflatoxin biosynthetic genes in these tissues. Interestingly, GUS activity was not observed in the hyphae colonizing the testa.     

Isolation of microsomal cytochrome-p450 isozymes from Ustilago maydis and their interaction with sterol demethylation inhibitors

A procedure for the isolation of microsomes containing cytochrome-P450 isozymes from Ustilago maydis is described. Yields of P450 amount to approximately 19(±+ 6) pmol mg^?1 of microsomal protein. The wavelength of maximum absorbance of the reduced carbon monoxide difference spectrum is 448-449 nm. The azole fungicides prochloraz, etaconazole, imazalil, triadimefon and 3-(2,4-dichlorophenyl)-2-(1H-1,2,4-triazol-1-yl)-4(3H)-quinazoline, which differ markedly in toxicity to U. maydis, all induce type II binding difference spectra at extremely low concentrations (10^?9-10^?8 M). The DMI concentrations which cause half saturation of type II binding difference spectra (IC₅₀) do not correlate with the fungicidal activities of the azoles. Binding of carbon monoxide to ferrous cytochrome-P450 was only slightly inhibited to different degrees by the DMIs tested. However, the inhibition of carbon monoxide binding also does not correlate with fungitoxicity of the DMIs. The results in this paper suggest that the spectrophotometric studies with this preparation are not useful for evaluating selective toxicity of DMIs to intact sporidia of U. maydis.     

Expression of <Emphasis Type="Italic">Xanthomonas oryzae </Emphasis>pv. <Emphasis Type="Italic">oryzae hrp</Emphasis> Genes in XOM2, a Novel Synthetic Medium

To analyze the regulation of hrp expression and to detect and identify hrp-dependent secretion proteins of plant-pathogenic bacteria, an appropriate hrp-inducing medium is indispensable. In this study, two efficient hrp-inducing media for Xanthomonas oryzae pv. oryzae were designed by assaying the expression of a hrcU (the first gene of the hrpC operon) and a gus (β-glucuronidase) fusion gene. We modified XVM2, which is a hrp-inducing medium for X. campestris pv. vesicatoria, by adding 0.01% xylose in place of fructose and sucrose (0.18 and 0.34%, respectively) as a sugar source. The resulting medium induced approximately 15-fold more GUS activity from transformants containing a hrcU::gus gene than did XVM2. Moreover, a methionine-containing synthetic medium with 0.18% xylose as a sugar source was able to induce much stronger expression of HrcU::GUS, with GUS activity approximately 100-fold greater than that in XVM2. Induction depended on a regulator, HrpXo, and the PIP (plant-inducible-promoter) box, suggesting that HrcU::GUS was expressed in a hrp-dependent manner. The induction of operons hrpA to hrpF in XOM2 was also confirmed. These results suggest that both media, especially XOM2, are highly efficient hrp-inducing media for X. oryzae pv. oryzae. Received 7 October 2002/ Accepted in revised form 22 November 2002     

The mechanism of resistance to sterol 14α demethylation inhibitors in a mutant (Erg 40) of Ustilago maydis

Resistance to DMI fungicides is a problem in both agriculture and medicine. Several mechanisms of resistance exist, but, as yet, few have been characterised in field resistant strains of plant pathogens. One approach to evaluating the role of mutations in the sterol 14α demethylase (14DM) target site requires cloning this gene and confirming its identity by complementation in an appropriate mutant. The azole‐resistant mutant, Erg 40, of Ustilago maydis which is totally blocked at the 14α demethylation step in sterol biosynthesis seems to be suitable for such expression studies. Transformation of Erg 40 with a plasmid containing the yeast 14α demethylase (CYP51A1) gene removed the block in sterol biosynthesis and generated azole‐sensitive transformants. Detailed analysis of these transformants failed to detect the presence of the yeast gene and suggested, instead, that changes in sterol biosynthesis resulted simply from the transformation protocol and not from the incorporation of extracellular DNA. Subsequent sequence analysis has revealed a mutation in the 14α demethylase gene of Erg 40. The results suggest that azole resistance in Erg 40 is not simply controlled by this mutation but involves some additional regulatory function, and consequently Erg 40 is not suitable for complementation studies with CYP51A1 genes. © 2000 Society of Chemical Industry     

Fungal derived cytokinins are necessary for normal Ustilago maydis infection of maize

Phytohormones derived from fungi play a key role in regulating plant–pathogen interactions; however, deciphering the separate contributions of pathogen and plant during infection has been difficult. Here, the Ustilago maydis–Zea mays pathosystem was used to investigate this chemical exchange. Ustilago maydis, the causative agent of maize smut, produces cytokinins (CK), which are a group of phytohormones responsible for directing plant development. The characteristic symptom of smut disease is the formation of tumours composed of plant and fungal tissue. Isopentenyltransferase (IPT) catalyses the rate‐limiting step in CK biosynthesis, and U. maydis strains in which the sole tRNA‐ipt gene was deleted no longer produced CKs. These deletion strains elicited fewer, smaller tumours than the pathogenic strain SG200. High performance liquid chromatography‐electrospray ionization tandem mass spectrometry (HPLC‐ESI MS/MS) was used to detect and quantify phytohormone levels in infected tissue. This revealed that key hormone changes in SG200 infections were not present in infections by deletion strains, suggesting that CK production by U. maydis is required for the altered phytohormone profile in infected tissue relative to uninfected tissue. Separate analyses indicated that U. maydis tRNA‐ipt mutants might be altered in their ability to metabolize CKs taken up from the environment. Mining the U. maydis genome identified genes encoding putative CK signalling and biosynthesis proteins.     

Genetic control of resistance to tridemorph inustilago maydis

Mutants ofUstilago maydis with low resistance to tridemorph isolated in a mutation frequency of 7x 10^-6 after UV-irradiation and selection on media containing 25 μg ml^-1 tridemorph. Genetic analysis with nine such mutant isolates resulted in the identification of two unlinked chromosomal loci,U/tdm- 1 andU/tdm- 2. TheU/tdm mutations are responsible for low resistance levels to tridemorph (resistance factor, Rf, of 3 or 5 based on effective concentration causing a 50% reduction in the growth rate (EC₅₀) or minimal inhibitory concentration (MIC) values, respectively) and low to moderate level of resistance to fenpropimorph (Rf 10 or 16 based on MIC or EC₅₀, respectively) and fenpropidin (Rf 5 or 11 based on MIC or EC₅₀, respectively). Haploid strains carrying bothU/tdm mutations exhibit higher levels of resistance to the above fungicides, indicating interallelic interaction between nonallelic genes. Crosses between mutants carrying theU/tdm- genes with compatible isolates carrying theU/fpm- 1 orU/fpm- 2 mutations, which were found in previous work to carry fenpropimorph resistance, yielded in all cases a large number of recombinants with wild-type sensitivity, indicating that the mutant genes involved were not allelic. Cross-resistance studies with the inhibitors of C-14 demethylase showed that. the U/tdm-mutations were responsible for increased sensitivity to the triazoles triadimefon, triadimenol, propiconazole and flusilazole, and to the pyridine pyrifenox. Study of gene effect on the fitness ofU. maydis showed thatU/tdm-mutations appeared to be pleiotropic, having more or less adverse effects on growth rate in liquid culture and pathogenicity on young corn plants.     

Conditional expression of harpinPsscauses yeast cell death that shares features of cell death pathway with harpinPss-mediated plant hypersensitive response (HR)

Conditional expression of harpin_Psscauses yeast cell death that shares features of cell death pathway with harpin_Pss-mediated plant hypersensitive response (HR).Pseudomonas syringae pv.syringae 61 hrp Z gene encodes harpin_Pss, a 34.7 kD extracellular protein that elicits a hypersensitive response (HR) in plants. Conditional expression of either full-length or truncated hrp Z sequences under the GAL1 promoter caused cell death in Saccharomyces cerevisiae Y187. Plating of pYEUT- hrp Z transformants on a medium containing galactose resulted in complete inhibition of colony formation, whereas their growth on a glucose-based medium was unaffected. Western blot analysis confirmed the expression of harpin_Pssin yeast cells transformed with pYEUT- hrp Z and grown in galactose-containing medium. A time-dependent decline in the percentage of trypan blue-excluding cells in cultures of pYEUT- hrp Z transformants was observed when cultured on galactose-containing medium. Similarly, the number of viable cells reduced to about 50% within 6 h. There were similarities in the harpin_Pss-mediated cell death in plants and yeast cell death (YCD). Galactose-induced cell death in pYEUT-hrp Z transformants of S. cerevisiae Y187 was suppressed by a protein kinase inhibitor K252a (10 μ M). The viability of pYEUT- hrp Z transformants was prolonged in the presence of 100 U ml⁻¹catalase suggesting a role for the oxidative burst in YCD that was further supported by the flow cytometric patterns of propidium iodide uptake by yeast cells. Overall, it appears that yeast provides a useful model system to understand the molecular mechanism of harpin_Pss-mediated cell death.     

Use of GUS Transformants of Fusarium subglutinans for Determining Etiology of Mango Malformation Disease

ABSTRACT Fusarium subglutinans has been associated with mango floral and vegetative malformation, although confusion exists regarding the etiology of the disease. A wild-type isolate of F. subglutinans causing mango malformation disease was transformed with the GUS (beta-glucuronidase) reporter and hygromycin resistance genes. Five stable transformants were isolated containing varying copy numbers at different integration sites. Specific GUS activity was quantified for the transformants, whereas no activity was recorded for the wild-type isolate. The transformants and the wild-type isolate were inoculated into healthy mango floral and vegetative buds. Typical symptoms of misshapen shoots with short internodes, stubby leaves, and bunchy, malformed inflorescences were observed 6 to 8 weeks following inoculation. The presence of GUS-stained mycelium of the pathogen viewed microscopically within infected plant organs provided unequivocal evidence that F. subglutinans is indeed a causal agent of mango malformation disease.     

向日葵黄萎病菌突变体库的建立及其阳性转化子的鉴定

为揭示向日葵大丽轮枝菌Verticillium dahliae Kleb.的致病机理,利用农杆菌介导法将带有潮霉素抗性标记和绿色荧光蛋白报告基因的双元载体转入大丽轮枝菌的分生孢子中并获得阳性转化子,以野生型菌株为对照,对随机挑取的阳性转化子的菌落形态、菌丝生长速率、产孢量、粗毒素分泌量和致病力进行了研究。结果表明,共获得800株阳性转化子,随机选取的40株阳性转化子中有2株的菌落只产生白色气生菌丝,不能形成黑色微菌核。与对照相比,40株转化子的生长速率均有不同程度降低,其中转化子A1生长速度降低最显著,菌落直径仅为3.28 cm,比对照下降了38.92%。40株转化子中有3株的产孢量高于对照,其中转化子A9的产孢量最高,为3.50×10~7个/mL,比对照提高1.10倍;转化子A1的产孢量最低,仅为1.35×10~7个/mL,比对照下降了19.16%。40株转化子中有4株的粗毒素分泌量较对照显著升高,占测定菌株的10%,有24株较对照显著降低,占60%,其余12株与对照无显著差异。40株转化子中有3株的致病力较对照显著增强,占测定菌株的7.5%;有7株的致病力较对照显著降低,占17.5%;其余30株与对照无显著差异。     

An Ulva armoricana extract protects plants against three powdery mildew pathogens

The protective activity of a crude extract prepared from the green macroalga, Ulva armoricana, previously shown to induce plant defence responses, was evaluated on three plant species, common bean, grapevine and cucumber, cultivated in the greenhouse and inoculated with three powdery mildew pathogens Erysiphe polygoni, E. necator and Sphareotheca fuliginea respectively. Chemical analyses showed that the extract was enriched in ulvans, which are green algae polysaccharides essentially composed of uronic acid and sulphated rhamnose. Weekly applications were performed by spraying of the green algal extract at various dilutions on bean, grapevine and cucumber leaves. A significant effect (50% protection) was observed using a dilution corresponding to about 3 g l⁻¹ dry matter and up to 90% reduction of symptom severity was obtained for the highest concentration (1/9 dilution, 6 g l⁻¹ dry matter) for the three plant species. To study the natural variability of the protective activity, five extracts prepared from algae batches harvested at different year periods were evaluated. Although polysaccharide composition varied among batches, all extracts elicit a reporter gene regulated by a defence-gene promoter in a transgenic tobacco line, and protect cucumber plants against powdery mildew infection. Together, these data demonstrate that U. armoricana is a reproducible source of active compounds which can be used to efficiently protect crop plants against powdery mildew diseases.