Identification of Resistance to Barley Leaf Stripe Using a Pyrenophora graminea Transformant Expressing β-glucuronidase

A Pyrenophora graminea strain expressing the -glucuronidase gene (GUS) was obtained via genetic transformation, and used to follow the penetration of the pathogen inside barley germinating seeds and the colonization of host tissues. Significant differences between resistant and susceptible barley cultivars were observed in the colonization of artificially-infected embryos by the fungus. These results suggest that the GUS transgenic strain of P. graminea will be useful for the early screening of barley cultivars for resistance to leaf stripe disease.     

小麦抗白粉病基因Pm6的微卫星标记鉴定

 Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici, is a prevalent disease worldwide. Breeding and planting resistance cultivars have been proved effective and environmental friendly for control of the disease. To develop easily used PCR-based markers in marker assisted selection (MAS) for Pm6, a dominant powdery mildew resistance gene in wheat, 25 microsatellite markers on chromosome 2BL in wheat were screened between susceptible parent Yumai13 and resistance parent Timgalen carrying Pm6. F₂ population derived from Yumai13 and Timgalen was further analyzed by the marker Xgwm501. The results indicated that Xgwm501 was a co-dominant marker linked to Pm6 gene at a distance of 14.8 cM. 29 Pm-carrying varieties were tested by the marker Xgwm501 and only those carrying Pm6 showed 117 bp resistance specific band. This marker is proved to have high practicability and can be used in MAS of Pm6 gene in wheat breeding programs.     

含双病原物诱导启动子植物安全表达载体的构建

 本研究用来自烟草的具有高度病原物特异性的两个诱导启动子EAS4和hsr203J,串联驱动GUS基因的表达,同时考虑到转基因植物的安全性,引入双边界序列,构建了含双病原物诱导启动子的植物安全表达载体。将表达载体转化烟草获得转基因植株。分析显示,在正常生长情况下转基因烟草检测不到GUS活性,或活性极低;而受疫霉激发子parasiticein、Phytophthora nicotianea[0]的孢子悬浮液和Ralstonia solanacarum的菌悬液诱导后,转基因烟草叶片中可检测到明显的GUS活性。结果表明,构建的植物表达载体所含双病原物诱导启动子均具有良好的诱导活性,可用于植物遗传转化。     

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。     

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     

Resistance to Oculimacula yallundae and Oculimacula acuformis is conferred by Pch2 in wheat

The recent report of a differential response of wheat lines containing the Pch2 gene to infection with the eyespot pathogens Oculimacula yallundae and O. acuformis has prompted this re‐examination of the response to these fungi by the recombinant lines used to map Pch2. Homozygous recombinant substitution lines (RSL) derived from the hybridization of Chinese Spring (CS) and the CS chromosome substitution line Cappelle Desprez 7A (CS/CD7A), previously evaluated for response to glucuronidase (GUS)‐transformed O. yallundae, were evaluated for response to infection with GUS‐transformed O. acuformis. Based on visual scores and on GUS expression level, which reflects fungal colonization of seedling plants, evidence of a quantitative trait locus (QTL) conferring resistance to O. acuformis was found in two separate growth chamber experiments (logarithm of the odds, LOD, = 2·7 and 6·7 at 305 and 289 cM, respectively) that was equivalent in location to that for resistance to O. yallundae (LOD = 13·2 and 11·4 at 289 and 304 cM, respectively). These results confirm that Pch2 confers some degree of resistance against both O. yallundae and O. acuformis under these conditions.     

Distribution and properties of geographically distinct isolates of sugar beet yellowing viruses

From a total of 261 yellow sugarbeet leaves collected from 10 countries representing three continents, the incidence and distribution of strains of Beet mild yellowing virus (BMYV), Beet chlorosis virus (BChV) and Beet yellows virus (BYV) were analysed using serological and molecular methods. BMYV was found in all countries except Greece, and more frequently in the northern and western areas of Europe, whereas BYV predominated in Turkey, Spain, Greece, the USA and Chile. BChV, originally found in the USA and the UK in 1989, was identified in France, Spain, the Netherlands and Chile. Nine sugar beet poleroviruses, plus a reference isolate of Turnip yellows virus (TuYV, syn. Beet western yellows virus ), were further characterized and compared. Isolates obtained from sugar beet infected this species, but not oilseed rape or lettuce; all isolates except one infected Capsella bursa-pastoris . The coat-protein sequences of these isolates were highly similar, with the consensus sequence representing 89% of nucleotide residues. Within the coat-protein gene, two regions were identified that could represent specific epitopes to which monoclonal antibody BYDV-PAV-IL-1 could bind; this antibody is used to distinguish beet poleroviruses in ELISA. Comparison of the sequences at the 5' end showed that sequence homology existed only between isolates with the same host range. The first sequence data of polerovirus isolates from Chile are presented, showing that the coat protein and the 5' end of their genomes are highly similar to those of BMYV isolates found in Europe. Chilean polerovirus isolates may have been imported from the northern hemisphere in sugar beet breeding material.     

Quantitative trait loci conferring blast resistance in hexaploid wheat at adult plant stage

Blast disease, caused by the Magnaporthe oryzae Triticum pathotype (MoT), is a major concern for wheat production in tropical and subtropical regions. The most destructive symptoms occur in wheat spikes. Infected spikes become bleached due to partial or total sterility, producing small and wrinkled grains. High disease pressure of the disease results in significant yield losses. This study aimed to identify wheat quantitative trait loci (QTLs) conferring resistance to blast disease at the heading stage. A doubled-haploid population was developed from the cross between BRS 209 (susceptible) and CBFusarium ENT014 (resistant, carrying the 2NS translocation). A linkage map was constructed containing 5,381 molecular markers and the inclusive composite interval mapping method was employed for QTL detection. Four QTLs were mapped in response to two MoT isolates. The major QTL identified on the 2AS chromosome explained an average of 84.0% of the phenotypic variation for spike bleaching at 9 days postinoculation and reinforces the potency of the 2NS translocation. Recombination between the distal region of chromosome 2AS and the 2NS marker was found. These results could explain why some lines carrying the VENTRIUP/LN2 marker have a variable reaction to the disease. QTLs on 5B and 7B chromosomes were also identified. Two mechanisms of resistance were hypothesized: the hypersensitive response and resistance to colonization of host tissues. The KASP markers thus developed and simple sequence repeats (SSRs) allocated in QTL regions can be used in the future for the development of wheat blast-resistant cultivars.     

Isolation and Characterization of a New Virulence Gene (abvA) of Agrobacterium tumefaciens

A mutant (M-1) was isolated by transposon (Tn5) insertion mutagenesis of Agrobacterium tumefaciens (strain A-208, C58 chromosome, nopaline type T37 pTi, virulent). The M-1 mutant exhibited a complete avirulent phenotype on Kalanchoe daigremontiana leaf and Kalanchoe pinnata stem but a very attenuated virulent phenotype on root of Daucus carota. The mutant had one insertion of Tn5 in pTi. A wild-type target segment (2.3 kb) that included the site of Tn5 insertion in M-1 mutant was cloned. Introducing the 2.3 kb segment into M-1 complemented completely the avirulent phenotype, producing galls as big as strain A-208. The 2.3 kb segment was sequenced, identifying three open reading frames, ORF 1 (354 bp), ORF 2 (261 bp) and ORF 3 (801 bp) in the segment. A Tn5 was inserted between the third and fourth nucleotide of ORF 1 in M-1. The ORF 1 had no homology to any reported genes and thus was named the abvA gene. The ORF 3 had the high homology (identities 44%, positive 68%) to the gene of the sarcosine oxidase β subunit (accession no. sp/P40875). Introduction of the DNA segment (743 bp) containing the abvA gene and its promoter region into M-1 partially complemented the avirulent phenotype of the mutant, producing galls smaller than strain A-208. The abvA gene was distributed not only on nopaline-type pTi (T37) but also on octopine-type pTi (A6NC) and chromosome (C58) of A. tumefaciens. M-1, being avirulent on K. daigremontiana and K. pinnata, had a Tn5 insertion only in the abvA gene on pTi but not in the abvA gene on the chromosome, implying that the abvA gene on the chromosome in strain A-208 is not functional. A binary vector, pIG121-Hm, containing the β -glucuronidase (GUS) gene with an intron was introduced into M-1, which was then applied to leaves of K. daigremontiana to assay GUS activity for monitoring T-DNA transfer to the host nucleus. High GUS activity comparable to that in strain A-208 was detected in M-1 in spite of its inability to induce galls, suggesting that M-1 can transfer T-DNA into the host nucleus, but cannot integrate it into the chromosome. Received 25 October 2000/ Accepted in revised form 28 December 2000     

Biological and sequence comparisons of Potato virus Y isolates associated with potato tuber necrotic ringspot disease

The biological and molecular relationships between a large number of Potato virus Y (PVY) isolates were examined, concentrating mainly on isolates associated with potato tuber necrotic ringspot disease (PTNRD). Following detailed analysis of the coat-protein gene, four main groups were identified which broadly corresponded to the phenotype of the different isolates. The groups comprised the ordinary strain (PVY^O), the necrotic strain (PVY^N), the C strain (PVY^C) and a group of recombinant (between ordinary and necrotic) isolates. In the latter group, all members were associated with PTNRD. However, four nonrecombinant isolates were also identified which were associated with PTNRD or tuber necrosis. Three were from tubers showing PTNRD symptoms in the field, while the fourth originated from symptomless tubers, but could cause necrotic rings on tubers under glasshouse conditions. The results show that although coat-protein recombination is always found associated with the PTNRD phenotype, some nonrecombinant isolates have very similar biological properties.     

Expression of the Arabidopsis MCM Gene PROLIFERA During Root-Knot and Cyst Nematode Infection

ABSTRACT Expression of the Arabidopsis thaliana gene PROLIFERA (PRL) was examined during development of root-knot and cyst nematode feeding sites. These obligate plant parasites establish specialized feeding structures in roots that allow them to withdraw nutrients from the host. In the process of establishing feeding sites, nematodes alter cell cycle regulation. PRL is normally expressed specifically in dividing cells at all stages of plant development and was used here as a marker for cell division. PRL expression, reported from a PRL::GUS fusion protein, was detected in nematode feeding sites of both root-knot and cyst nematodes from the earliest stages of infection in both giant cells and syncytia. However, unlike other cell cycle genes, expression of PRL was detected only occasionally in cells surrounding the feeding sites. PRL::GUS activity persisted until late in the infection cycle, past the time when other cell cycle genes are expressed. These data indicate that some aspects of the PRL expression pattern during nematode infection differ from that of other cell cycle genes.     

Macroscopic and Histological Characterisation of Genes er1 and er2 for Powdery Mildew Resistance in Pea

In pea, two single recessive genes, er1 and er2, have been identified for resistance to powdery mildew caused by Erysiphe pisi, but little is known about their mode of action. Pea accessions carrying the genes er1 or er2 and other accessions displaying resistance to powdery mildew in the field were studied. In accessions carrying gene er1, epidermal cell penetration was prevented and very few haustoria or colonies were formed. Under controlled conditions, er1 conferred complete or almost complete resistance to the fungal isolates used and this resistance was not associated with macroscopically visible necrosis. Under field conditions these accessions developed a low level of disease. Resistance in line JI2480 (carrying er2) increased with temperature and leaf age, and complete resistance was expressed only at high temperature (25 °C) or in mature leaves. This resistance was based mainly on post-penetration cell death, complemented by a reduction of percentage penetration success in mature leaves. Combining the resistance provided by gene er1 and by line JI2480 into new cultivars is likely to increase their level of resistance and enhance durability of the protection.     

<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation for random insertional mutagenesis in <Emphasis Type="Italic">Colletotrichum lagenarium</Emphasis>

Random insertional mutagenesis using a marker DNA fragment is an effective method for identifying fungal genes relevant to morphogenesis, metabolism, and so on. Agrobacterium tumefaciens-mediated transformation (AtMT) has long been used as a tool for the genetic modification of a wide range of plant species. Recent study has indicated that A. tumefaciens could transfer T-DNA not only to plant cells but also to fungal cells. In this study, AtMT was applied to Colletotrichum lagenarium for random insertional mutagenesis. We constructed a binary vector pBIG2RHPH2 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of C. lagenarium wild-type 104-T with pBIG2RHPH2-introduced A. tumefaciens C58C1 led to the production of 150–300 hygromycin-resistant transformants per 10⁶ conidia. Southern blot analysis revealed that T-DNA was mainly integrated at a single site in the genome and at different sites in transformants. The T-DNA inserts showed small truncations of either end, but the hygromycin-resistant gene cassette inside the T-DNA was generally intact. The mode of T-DNA insertion described above resulted in highly efficient gene recovery from the transformants by thermal asymmetrical interlaced-polymerase chain reaction. The fungal genomic DNA segments flanking T-DNA were identified from five of eight mutants that had defective melanin biosynthesis. The sequence from one of the segments was identical to that of the melanin biosynthesis gene PKS1 of C. lagenarium, which we previously characterized. These results strongly support our notion that AtMT is a possible tool for tagging genes relevant to pathogenicity in the plant pathogenic fungus C. lagenarium.     

NPTⅡ基因和GUS基因在小麦遗传转化中的应用

对小麦遗传转化上常用的选择标记基因NPTⅡ的选择剂及GUS基因在幼胚愈伤组织中的转移进行了研究。结果表明用G418作为选择剂,浓度为20-30mg／L较为合适。幼胚愈伤组织与农杆菌共培养3d后,即可检测到GUS基因的瞬时表达。农杆菌LBA4404由500 mg／L羧卞青霉素抑制;EHA105、AGLI由500 mg／L头孢霉素抑制。而不能用卡那霉素。     

Elucidation of Septoria tritici x Wheat Interactions Using GUS-Expressing Isolates

ABSTRACT Isolate ISR398 of Septoria tritici (which produces none to few pycnidia on the wheat cv. Seri 82 and high coverage on cv. Shafir) and isolate ISR8036 (which is virulent on both cultivars) were genetically cotrans-formed using the selectable marker gene hph, which confers resistance to hygromycin B (hygB), and the reporter gene uidA, encoding beta-glucuronidase (GUS). Most of the genetically transformed isolates (98.8%) produced similar pycnidial coverage on seedlings of 'Seri 82' and 'Shafir' as the two wild-type isolates. Southern analysis of 25 randomly selected hygB(R)GUS(+) transformants probed with the uidA sequence revealed multiple insertion sites. GUS activity was determined fluorimetrically by measuring the conversion of 4-methylumbelliferyl beta-D-glucuronide (MUG) to 4-methylumbelliferone (MU). The high GUS-expressing transformants 398D97 and 8036E27 were used to elucidate fungal development within inoculated leaf tissue by using GUS activity to estimate the fungal proteins content in planta. Increase in fungal biomass was recorded in 'Shafir' inoculated with the GUS-expressing transformants 398D97 and 8036E27 following a 12-day latent period. A 15-day latent period was recorded in 'Seri 82' inoculated with 8036E27, whereas an 18-day latent period was recorded on 'Seri 82' inoculated with 398D97 and the two mixtures 398D97 + ISR8036 and ISR398 + 8036E27. The rate of fungal development and the estimated level of fungal proteins at the pycnidia maturation stage was high in leaves of 'Shafir' and moderate to low on 'Seri 82', even in cases in which no significant differences were recorded in pycnidial coverage. An endogenous capacity to hydrolyze beta-1,4-D-glucuronidase was recorded in leaves inoculated with wild-type isolates. The latent periods in MU production of the uidA-expressing transformants mimicked those recorded for the wild-type isolates. However, at all stages, the levels of MU produced in wheat inoculated with wild-type isolates were markedly lower than those produced by GUS-expressing transformants. The mode of interaction (compatible or incompatible) determined the onset of the induction, rate, and level of enzyme production.     

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.