Identification of a new virulence locus in Agrobacterium tumefaciens that affects polysaccharide composition and plant cell attachment.

We have identified a new virulence locus in Agrobacterium tumefaciens. Strains carrying Tn5 inserts at this locus could not incite tumors on Kalanchoe daigremontiana, Nicotiana rustica, tobacco, or sunflower and had severely attenuated virulence on carrot disks. We termed the locus pscA, because the mutants that defined the locus were initially isolated as having an altered polysaccharide composition; they were nonfluorescent on media containing Leucophor or Calcofluor, indicating a defect in the production of cellulose fibrils. Further analysis showed that the pscA mutants produced little, if any, of the four species of exopolysaccharide synthesized by the wild-type strain. DNA hybridization analysis and genetic complementation experiments indicated that the pscA locus is not encoded by the Ti plasmid and that it is distinct from the previously described chromosomal virulence loci chvA and chvB. However, like chvA and chvB mutants, the inability of the pscA mutants to form tumors is apparently due to a defect in plant cell attachment. Whereas we could demonstrate binding of the wild-type strain to tobacco suspension cells, attachment of the pscA mutants was drastically reduced or completely absent.     

Agrobacterium rhizogenes mutants that fail to bind to plant cells.

Transposon insertion mutants of Agrobacterium rhizogenes were screened to obtain mutant bacteria that failed to bind to carrot suspension culture cells. A light microscope binding assay was used. The bacterial isolates that were reduced in binding to carrot cells were all avirulent on Bryophyllum diagremontiana leaves and on carrot root disks. The mutants did not appear to be altered in cellulose production. The composition of the medium affected the ability of the parent and mutant bacteria to bind to carrot cells. The parent strain bound to carrot cells in greatest numbers in low-ionic-strength media such as 4% sucrose but still showed significant binding in Murashige-Skoog tissue culture medium. All of the mutants showed reduced binding in 4% sucrose after 2 h of incubation with carrot cells. One mutant was delayed in binding in 4% sucrose. This mutant and one other mutant also showed reduced binding to carrot cells in Murashige-Skoog medium. To determine whether the Tn5 insertion was responsible for the mutant phenotype, DNA containing the Tn5 insertion was cloned from the mutant bacteria and used to introduce Tn5 into the parent strain in the same location as in the original mutant by marker exchange. The resulting transconjugants had the same avirulent, nonattaching phenotype as the original mutants, suggesting that the mutant phenotype was due to the Tn5 insertion. The cloned DNA containing the Tn5 insertion was also tested for homology to DNA of known genes that affect attachment of Agrobacterium tumefaciens to plant cells by DNA hybridization. No homology to chv, att, or pscA clones was observed. In addition, cloned chv, att, and pscA genes from A. tumefaciens were unable to complement the attachment-minus A. rhizogenes mutants. Thus, the A. rhizogenes nonattaching mutants appear to be different from the previously described A. tumefaciens mutants.     

Agrobacterium tumefaciens virulence locus pscA is related to the Rhizobium meliloti exoC locus.

Agrobacterium tumefaciens and Rhizobium meliloti carry related genetic loci which have important roles in virulence and symbiosis. Previously, it was shown that two virulence loci of A. tumefaciens, chvA and chvB, are related to two R. meliloti symbiosis loci, ndvA and ndvB, respectively (T. Dylan, L. Ielpi, S. Stanfield, L. Kashyap, C. Douglas, M. Yanofsky, E. Nester, D. R. Helinski, and G. Ditta, Proc. Natl. Acad. Sci. USA 83:4403-4407, 1986). Here we show that these two phytobacteria possess additional related virulence/symbiosis genes. Results of genetic complementation and DNA hybridization experiments indicate that the pscA virulence locus of A. tumefaciens is structurally and functionally related to the exoC symbiosis locus of R. meliloti. Thus, A. tumefaciens and R. meliloti bear at least three related genetic loci that have crucial roles in establishing the interactions that each bacterium has with its respective host plants.     

基于TAC载体的水稻转化系统的建立

将含有大约50kb水稻基因组片段的TAC17克隆(NK15)通过电击转化到农杆菌LBA4404中,经多次继代培养,该克隆在农杆菌中是稳定的。用常规的农杆菌介导方法将该克隆转化粳稻品种农垦58S成熟胚的愈伤组织,对T0代进行PCR和Southern杂交分析表明,TAC17所携带的50kb外源DNA片段已完整地整合到水稻基因组上,整合方式多数为单位点插入,整合位点是随机的。经T1代分析表明,外源基因可以稳定地遗传,而且进一步确定外源大片段的整合方式为为单位点插入。     

Common loci for Agrobacterium tumefaciens and Rhizobium meliloti exopolysaccharide synthesis and their roles in plant interactions.

Mutants of Rhizobium meliloti have been isolated which are deficient in exopolysaccharide (EPS) production and effective nodulation of alfalfa (J. A. Leigh, E. R. Signer, and G. C. Walker, Proc. Natl. Acad. Sci. USA 82:6231-6235, 1985). We isolated approximately 100 analogous EPS-deficient (Exo) mutants of the closely related plant pathogen Agrobacterium tumefaciens, including strains whose EPS deficiencies were specifically complemented by each of five cloned R. meliloti exo loci. We also cloned A. tumefaciens genes which complemented EPS defects in three of the R. meliloti Exo mutants. In two of these cases, symbiotic defects were also complemented. All of the A. tumefaciens Exo mutants formed normal crown gall tumors on four different plant hosts, except ExoC mutants, which were nontumorigenic and unable to attach to plant cells in vitro. Like their R. meliloti counterparts, A. tumefaciens Exo mutants were deficient in production of succinoglycan, the major acidic EPS species produced by both genera. A. tumefaciens ExoC mutants also produced extremely low levels of another major EPS, cyclic 1,2-beta-D-glucan. This deficiency has been noted previously in a different set of nontumorigenic, attachment-defective A. tumefaciens mutants.     

Bacterial transposons are co-transferred with T-DNA to rice chromosomes during Agrobacterium-mediated transformation

Agrobacterium tumefaciens is widely utilized for delivering a foreign gene into a plant's genome. We found the bacterial transposon Tn5393 in transgenic rice plants. Analysis of the flanking sequences of the transferred-DNA (T-DNA) identified that a portion of the Tn5393 sequence was present immediately next to the end of the T-DNA. Because this transposon was present in A. tumefaciens strain LBA4404, but not in EHA105 and GV3101, our findings indicated that Tn5393 was transferred from LBA4404 into the rice genome during the transformation process. We also noted that another bacterial transposon, Tn5563, is present in transgenic plants. Analyses of 331 transgenic lines revealed that 26.0% carried Tn5393 and 2.1% contained Tn5563. In most of the lines, an intact transposon was integrated into the T-DNA and transferred to the rice chromosome. More than one copy of T-DNA was introduced into the plants, often at a single locus. This resulted in T-DNA repeats of normal and transposon-carrying TDNA that generated deletions of a portion of the T-DNA, joining the T-DNA end to the bacterial transposon. Based on these data, we suggest that one should carefully select the appropriate Agrobacterium strain to avoid undesirable transformation of such sequences.     

大豆基因型对根癌农杆菌菌株敏感性的研究

以栽培大豆［Glycine max (L.) Mer］吉林30、吉林43、绥农8、黑农35和东农42等的下胚轴为外植体,用EHA105和LBA4404 2个根癌农杆菌菌株（分别含有pGBI121S4ABC和pGBI4A2B质粒）研究大豆基因型对根癌农杆菌的敏感性,以及根癌农杆菌对大豆的侵染能力。结果表明,大豆基因型对根癌农杆菌的敏感性存在显著差异,以吉林43最敏感。根癌农杆菌菌株对大豆下胚轴侵染能力不同,含有pGBI121S4ABC质粒的LBA4404侵染能力较强,但差异未达显著水平。 Abstract:The sensitivity of genotypes in soybean to lines of Agrobacterium tumefaciens and the ability of A.tumefaciens infecting to soybean were investigated with hypocotyls of soybean (Jilin30,Jilin43,Suinong8,Heinong35 and Dongnong42) and lines of A.tumefaciens LBA4404 and EHA105 which including plasmid pGBI121S4ABC and pGBI4A2B respectively.The results showed that the sensitivity of genotypes in soybean to A.tumefaciens was significantly different.Jilin43 was the most sensitive materials to A.tumefaciens.The ability of A.tumefaciens infecting hypocotyls in soybean was different.LBA4404 including plasmid pGBI121S4ABC was easier to infect hypocotyls of soybean.     

PSAG12-ipt嵌合基因转化马铃薯体系的研究

以根癌农杆菌介导法将PSAG12-ipt嵌合基因导入马铃薯栽培品种,对影响马铃薯遗传转化的多种因素进行系统研究.结果表明:马铃薯茎段分化效率高于叶片,马铃薯愈伤诱导和芽分化最适培养基为MS+6-BA 0.25mg/L+NAA 0.25mg/L+2,4-D 0.25mg/L,添加1%Na2SO3能有效防止褐化;茎段愈伤诱导和分化苗生根最适的Kan浓度分别为50mg/L和75mg/L;外植体预培养2d,OD600为0.2～0.5的农杆菌浓度侵染8min、共培养3d后进行选择培养能有效地提高植株再生能力.用PSAG12和ipt双重PCR检测再生植株,阳性转化率为65.8%.Southern blotting结果表明,转基因植株多以单拷贝形式整合进马铃薯基因组中.     

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.     

金鱼草基因转化和转基因植株再生

本实验采用根癌农杆菌ＬＢＡ４４０４（ｐ３５ＳＧＵＳＩＮＴ与金鱼草下胚轴切段共培养,将ＧＵＳ基因导入金鱼草细胞,通过不定芽发生途径获得抗Ｇ４１８再生植株．经ＤＮＡ／ＤＮＡ斑点杂交及ＧＵＳ活性原位组织检测初步证实外源基因ＧＵＳ已整合进金鱼草基因组并得到表达．     

T-DNA locus structure in a large population of soybean plants transformed using the Agrobacterium-mediated cotyledonary-node method

Designing transformation experiments for either functional genomics or crop improvement requires knowledge of the transgene locus structure, number, transmission and expression resulting from a specific transformation method. We recently reported an improvement to the soybean [Glycine max (L.) Merrill] cotyledonary-node transformation method that resulted in the efficient production of transgenic plants. To characterize the transgene loci resulting from this method, we analysed 270 independent T0 plants and 95 randomly selected T1 progenies for T-DNA locus complexity using Southern analysis. The lines were transformed with Agrobacterium tumefaciens strains LBA4404 or EHA105 carrying the binary plasmids pGPTV, pTOK233, pCAMBIA1303 or pCAMBIA1309, and regenerated in medium supplemented with or without silver nitrate (AgNO3). Analysis in the T0 generation showed that the number of hpt-hybridizing fragments per plant ranged from 1-15, with 31.5% of the lines having a single hpt-hybridizing fragment. Each primary soybean transformant had, on average, 2.0 unlinked transgene loci and that half of the segregating loci in the T1 progenies were single, simple T-DNA insertions. Of the loci containing multiple T-DNA fragments, a low frequency had tandem and inverted repeat T-DNA structures. Integration of binary plasmid backbone sequences occurred in 37% of primary transformants. A. tumefaciens strain, binary plasmid and thiol treatment had no significant effect on transgene locus structure, numbers or expression. Interestingly, exposure of soybean explants to AgNO3 throughout shoot induction and elongation increased T-DNA locus complexity in the primary transformants and decreased silencing of gusA expression in the T1 generation.     

Stable genetic transformation of Eschscholzia californica expressing synthetic green fluorescent proteins

An efficient protocol is described for the stable genetic transformation of Eschscholzia californica (California poppy) using Agrobacterium tumefaciens as a vector. We have employed the disarmed A. tumefaciens LBA4404 encoding a synthetic green fluorescent protein reporter gene that is further controlled by an enhanced cauliflower mosaic virus 35S promoter. Stably transformed E. californica cells appear 3 weeks after initial cocultivation of A. tumefaciens with poppy leaves, stems, or roots. Transformed poppy calli were visualized by exposure to long-wave UV or blue light and analyzed in detail by fluorescent microscopy and laser-scanning microscopy. Moreover, green fluorescent calli have been maintained through continual subculture and grow well either on Gamborg's B5 agarose or liquid medium.     

Genetic transformation of barley (Hordeum vulgare L.) via infection of androgenetic pollen cultures with Agrobacterium tumefaciens

A novel genetic transformation method for barley (Hordeum vulgare L.), based on infection of androgenetic pollen cultures with Agrobacterium tumefaciens, is presented. Winter-type barley cv. 'Igri' was amenable to stable integration of transgenes mediated by A. tumefaciens strain LBA4404 harbouring a vector system that confers hypervirulence, or by the non-hypervirulent strain GV3101 with a standard binary vector. The efficacy of gene transfer was substantially influenced by pollen pre-culture time, choice of Agrobacterium strain and vector system, Agrobacterium population density, medium pH and the concentrations of acetosyringone, CaCl(2) and glutamine. After co-culture, rapid removal of viable agrobacteria was crucial for subsequent development of the pollen culture. To this end, the growth of agrobacteria was suppressed by the concerted effects of appropriate antibiotics, low pH, reduced level of glutamine and high concentrations of CaCl(2) and acetosyringone. Following infection with LBA4404 and GV3101, about 31% and 69%, respectively, of the primary transgenic (T(0)) plants carried a single copy of the sequence integrated. The use of hypervirulent A. tumefaciens and hygromycin resistance as a selectable marker resulted in 3.7 T(0) plants per donor spike. About 60% of the primary transgenic plants set seed, indicating spontaneous genome doubling. An analysis of 20 T(1) populations revealed that four progenies did not segregate for reporter gene expression. This indicates that the approach pursued enables the generation of instantly homozygous primary transgenic plants. The method established will be a valuable tool in functional genomics as well as for the biotechnological improvement of barley.     

Biochemical characterization of avirulent exoC mutants of Agrobacterium tumefaciens.

The synthesis of periplasmic beta(1-2)glucan is required for crown gall tumor formation by Agrobacterium tumefaciens and for effective nodulation of alfalfa by Rhizobium meliloti. The exoC (pscA) gene is required for this synthesis by both bacteria as well as for the synthesis of capsular polysaccharide and normal lipopolysaccharide. We tested the possibility that the pleiotropic ExoC phenotype is due to a defect in the synthesis of an intermediate common to several polysaccharide biosynthetic pathways. Cytoplasmic extracts from wild-type A. tumefaciens and from exoC mutants of A. tumefaciens containing a cloned wild-type exoC gene synthesized in vitro UDP-glucose from glucose, glucose 1-phosphate, and glucose 6-phosphate. Extracts from exoC mutants synthesized UDP-glucose from glucose 1-phosphate but not from glucose or glucose 6-phosphate. Membranes from exoC mutant cells synthesized beta(1-2)glucan in vitro when exogenous UDP-glucose was added and contained the 235-kilodalton protein, which has been shown to carry out this synthesis in wild-type cells. We conclude that the inability of exoC mutants to synthesize beta(1-2)glucan is due to a deficiency in the activity of the enzyme phosphoglucomutase (EC 2.7.5.1), which in wild-type bacteria converts glucose 6-phosphate to glucose 1-phosphate, an intermediate in the synthesis of UDP-glucose. This interpretation can account for all of the deficiencies in polysaccharide synthesis which have been observed in these mutants.     

抗生素对农杆菌的抑制和对油菜外植体分化的影响

通过哌拉青霉素、氨苄青霉素、青霉素钠、羧苄青霉素、头孢拉定、头孢唑啉钠、磷霉素钠、乳糖酸红霉素、白霉素9种抗生素对根癌农杆菌EHA105和LBA4404的抑制效果,以及对油菜子叶柄分化影响的研究,结果表明：羧苄青霉素在500mg／L时对农杆菌EHA105的抑菌效果最好,而其它8种抗生素对EHA105无明显的抑菌作用;对于LBA4404浓度为200mg／L的羧苄青霉素、氨苄青霉素和头孢唑啉钠都有良好的抑菌效果。不同抗生素对油菜子叶柄的分化试验结果表明,羧苄青霉素和头孢唑啉钠对离体油菜子叶柄再生分化及生长没有影响,磷霉素钠、乳糖酸红霉素、自霉素几乎完全抑制了油菜子叶柄分化。同时对卡那霉素(作为筛选剂)的浓度进行了筛选,确定了油菜33B的筛选浓度为15mg／L,油菜918B的筛选浓度为10mg／L。     

农杆菌介导将Bt杀虫蛋白基因导入优良玉米自交系的研究

以杂交育种中广泛使用的优良玉米自交系340、4112为材料,用带有质粒pGBIL04（Pactin-Bt-Tnos）的根癌农杆菌LBA4404转化其幼胚及其初始愈伤组织,共培养3天后,在含PPT的培养基上连续筛选培养3代,然后分化获得再生植株。PCR检测证明目的基因已整合到再生植株的基因组中。实验结果表明幼胚预培养后形成的新鲜的初始愈伤组织是比较适宜的转化受体,结果还发现将共培养温度降到22℃可以提高农杆菌介导的玉米遗传转化的筛选频率。 Abstract:Excellent inbred-lines of maize,340 and 4112,which were used largely in hybridized combination were transformed with Agrobacterium tumefaciens.The immature embryos and their original calli were infected by A.tumefaciens LBA4404 containing plasmid pGBIL04.After 3 days of co-cultivation,the immature embryos and calli were continuously selected on the medium containing phosphinothricin (PPT) for 3 generations,then plants were regenerated.It was proved by PCR analysis that the target Bt gene had been integrated into the genome of regenerated plants.The results showed that fresh original calli from the immature embryos after pre-culture were suitable acceptors.The results also showed that it could increase the frequency of selection by properly lowering the co-culture temperature to 22℃.     

菠菜甜菜碱醛脱氢酶基因在棉花中的过量表达和抗冻耐逆性分析

分离出菠菜甜菜碱醛脱氢酶基因(SoBADH)构建成由CaMV35S驱动的双元植物表达载体pBSB, 农杆菌菌株LBA4404携带该载体转化棉花, 获得转基因棉花植株。65株转基因植株经过PCR筛选、Southern blotting分析证明有45株为成功的转化株, 外源基因已经被整合到棉花的染色体组中, 并以单拷贝插入居多。对部分株系的SoBADH基因的表达进行分析表明均有较高的mRNA和蛋白的表达。经测定这些株系中的甜菜碱脱氢酶活性显著提高, 达到0.66~1.70 nmol/min/mg水平。同时这些转基因株系在盐胁迫下比对照长势强, 株高和地上部分的鲜重显著高于非转基因对照; 在低温胁迫下, 这些转基因株系表现出显著的抗冻性能。结果表明菠菜甜菜碱醛脱氢酶能够在异源植物棉花中过量表达, 并具有较高的酶活性, 转基因棉花可作为抗逆育种的种质材料。