大麦与白粉病菌互作中钙调素的细胞化学定位

对不同抗病性的大麦近等基因系受白粉病菌侵染诱导后的钙调素(CaM)变化进行了免疫细胞化学研究。结果表明,钙调素普遍存在于各种不同类型的细胞中,代谢活跃的细胞中CaM标记密度相对较高;CaM分布于细胞核、细胞壁、叶绿体等细胞器中,其中细胞核标记密度最高。在健康叶片中,感病品系Ingrid伴胞中CaM的标记密度明显高于抗病品系mlo—3,而其他细胞中CaM标记密度没有明显差别。病原菌接种后,各种细胞的CaM标记密度均呈现一定程度的上升,但Ingrid上升幅度相对较大。在叶肉细胞中,mlo-3细胞核中CaM增加最明显。并对叶肉细胞和伴胞中CaM的变化进行了讨论。     

Methoxylated fatty acids in Blumeria graminis conidia

The total fatty acids (FA) composition of Blumeria graminis f.sp. tritici conidia, the causal agent of wheat powdery mildew, was analyzed as a function of their age. A total of 19 FA (C12-C24 saturated and unsaturated) and unusual methoxylated fatty acids (mFA) were detected in young, intermediate and old conidia. Two very long chain methoxylated FA were identified by GC-MS as 3-methoxydocosanoic and 3-methoxytetracosanoic acids. Medium chain FA were predominant in young conidia (75%, including 13% of mFA) while very long chain fatty acids constituted the major compounds in old conidia (74%, including 30% of mFA). We have shown for the first time that the total FA composition is strongly correlated with the age of B. graminis f.sp. tritici (Bgt) conidia.     

Phytotoxic Nep1-like proteins from the necrotrophic fungus Botrytis cinerea associate with membranes and the nucleus of plant cells

Nep1-like proteins (NLPs), produced by an array of unrelated microorganisms, are phytotoxic for dicotyledonous plant cells but their mode of action has not yet been established. Two paralogous NLPs from the necrotrophic plant pathogenic fungus Botrytis cinerea were characterized, designated BcNEP1 and BcNEP2. Both proteins were produced in the heterologous host Pichia pastoris and purified to homogeneity. The localization of fluorescently labelled proteins was studied and mechanisms of cell death were investigated in protoplasts and suspension cells. Purified BcNEP1 and BcNEP2 caused necrosis in all dicotyledonous plant species tested, but not in monocotyledons. A synthetic heptapeptide comprising a sequence (GHRHDWE) that is conserved in all NLPs did not cause symptoms and was unable to interfere with necrosis induction by BcNEP1 and BcNEP2 proteins. Fluorescently labelled BcNEP1 and BcNEP2 proteins were associated with plasma membranes and the nuclear envelope, as well as in the nucleolus of responding plant cells. A strong hydrogen peroxide (H(2)O(2)) accumulation was observed in chloroplasts. The death process was characterized by TUNEL assays as apoptosis, necrosis or intermediate forms of both. BcNEP1- and BcNEP2-induced cell death execution could not be abolished by specific inhibitors. These results provide further information on mechanisms of NLP-inflicted cell death.     

Gene identification in the obligate fungal pathogen Blumeria graminis by expressed sequence tag analysis

Powdery mildew of barley is caused by the obligate fungal pathogen Blumeria graminis f. sp. hordei. Haploid conidia of B. graminis, landing on the barley leaf, germinate to form first a primary germ tube and then an appressorial germ tube. The appressorial germ tube differentiates into a mature appressorium from which direct penetration of host epidermis occurs. Here we present data on 4908 expressed sequence tags obtained from B. graminis conidia. The combined sequences represent 2676 clones describing 1669 individual genes. Comparison with sequences from other pathogenic and nonpathogenic fungi defines hypotheses on the genes required for pathogenicity and growth on the host. The putative roles of some of the identified genes are discussed.     

Interaction of a Blumeria graminis f. sp. hordei effector candidate with a barley ARF‐GAP suggests that host vesicle trafficking is a fungal pathogenicity target

Filamentous phytopathogens, such as fungi and oomycetes, secrete effector proteins to establish successful interactions with their plant hosts. In contrast with oomycetes, little is known about effector functions in true fungi. We used a bioinformatics pipeline to identify Blumeria effector candidates (BECs) from the obligate biotrophic barley powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh). BEC1–BEC5 are expressed at different time points during barley infection. BEC1, BEC2 and BEC4 have orthologues in the Arabidopsis thaliana‐infecting powdery mildew fungus Golovinomyces orontii. Arabidopsis lines stably expressing the G. orontii BEC2 orthologue, GoEC2, are more susceptible to infection with the non‐adapted fungus Erysiphe pisi, suggesting that GoEC2 contributes to powdery mildew virulence. For BEC3 and BEC4, we identified thiopurine methyltransferase, a ubiquitin‐conjugating enzyme, and an ADP ribosylation factor‐GTPase‐activating protein (ARF‐GAP) as potential host targets. Arabidopsis knockout lines of the respective HvARF‐GAP orthologue (AtAGD5) allowed higher entry levels of E. pisi, but exhibited elevated resistance to the oomycete Hyaloperonospora arabidopsidis. We hypothesize that ARF‐GAP proteins are conserved targets of powdery and downy mildew effectors, and we speculate that BEC4 might interfere with defence‐associated host vesicle trafficking.     

Location and mapping of the powdery mildew resistance gene MlRE and detection of a resistance QTL by bulked segregant analysis (BSA) with microsatellites in wheat

Powdery mildew (Blumeria graminis f. sp. tritici) is one of the most damaging diseases of wheat (Triticum aestivum). The objective of this study was to locate and map a recently identified powdery mildew resistance gene, MlRE, carried by the resistant line RE714 using microsatellites uniformly distributed among the whole genome together with a bulked segregant analysis (BSA). The bulks consisted of individuals with an extreme phenotype taken from a population of 140 F₃ families issued from the cross between RE714 (resistant) and Hardi (susceptible). The population had been tested with three powdery mildew isolates at the seedling stage. Qualitative interpretation of the resistance tests located the MlRE gene on the distal part of the long arm of chromosome 6A. A subsequent quantitative interpretation of the resistance permitted us to detect another resistance factor on a linkage group assigned to chromosome 5D, which was constructed with microsatellites for which a polymorphism of intensity between bulks was observed. This quantitative trait locus (QTL) explained 16.8– 25.34% of the total variation. An interaction between both the resistant factor (MlRE and the QTL) was found for only one of the isolates tested. This study shows the advantage of making a quantitative interpretation of resistant tests and that the use of microsatellites combined with BSA is a powerful strategy to locate resistance genes in wheat. Received: 30 August 1999 / Accepted: 11 November 1999     

Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea

Aims: FIP‐gsi, a fungal immunomodulatory protein found in Ganoderma sinense, has antitumour, anti‐allergy and immunomodulatory activities and is regulated by the fip‐gsi gene. In this study, we aimed to express the fip‐gsi gene from G. sinense in Coprinopsis cinerea to increase yield of FIPs‐gsi. Methods and Results: A fungal expression vector pBfip‐gsi containing the gpd promoter from Agaricus bisporus and the fip‐gsi gene from the G. sinense was constructed and transformed into C. cinerea. PCR and Southern blotting analysis verified the successful integration of the exogenous gene fip‐gsi into the genome of C. cinerea. RT‐PCR and Northern blotting analysis confirmed that the fip‐gsi gene was transcribed in C. cinerea. The yield of the FIP‐gsi protein reached 314 mg kg^?1 fresh mycelia. The molecular weight of the FIP‐gsi was 13 kDa, and the FIP‐gsi was capable of hemagglutinating mouse red blood cells, but no such activity was observed towards human red blood cells in vitro. Conclusions: The fip‐gsi from G. sinense has been successfully translated in C. cinerea, and the yield of bioactive FIP‐gsi protein was high. Significance and Impact of the Study: This is the first report using the C. cinerea for the heterologous expression of FIP‐gsi protein and it might supply a basis for large‐scale production of the protein.     

The biosynthetic pathway to abscisic acid via ionylideneethane in the fungus Botrytis cinerea

The biosynthetic pathway to abscisic acid (ABA) from isopentenyl diphosphate in the fungus, Botrytis cinerea, was investigated. Labeling experiments with (18)O2 and H2(18)O indicated that all oxygen atoms at C-1, -1, -1' and -4' of ABA were derived from molecular oxygen, and not from water. This finding was inconsistent not only with the known carotenoid pathway via oxidative cleavage of carotenoids, but also with the classical direct pathway via cyclization of farnesyl diphosphate. The fungus produced new C15-compounds, 2E,4E-alpha-ionylideneethane and 2Z,4E-alpha-ionylideneethane, along with 2E,4E,6E-allofarnesene and 2Z,4E,6E-allofarnesene, but did not apparently produce carotenoids except for a trace of phytoene. The C15-compounds labeled with 13C were converted to ABA by the fungus, and the incorporation ratio of 2Z,4E-alpha-ionylideneethane was higher than that of 2E,4E-alpha-ionylideneethane. From these results, it was concluded that farnesyl diphosphate was reduced at C-1, desaturated at C-4, and isomerized at C-2 to form 2Z,4E,6E-allofarnesene before being cyclized to 2Z,4E-alpha-ionylideneethane; the ionylideneethane was then oxidized to ABA with molecular oxygen. This direct pathway via ionylideneethane means that the biosynthetic pathway to fungal ABA, not only before but also after isopentenyl diphosphate, differs from that to ABA in plants, since plant ABA is biosynthesized using the non-mevalonate and carotenoid pathways.     

Competition Alters Temporal Dynamics of Sporulation in the Wheat Stem Rust Fungus

To investigate the effects of competition on the timing of pathogen reproduction, urediniospores of two strains of Puccinia graminis f.sp. tritici (SR22 and SR41) were inoculated onto leaves of wheat seedlings singly and in 1 : 1 mixture at three inoculum densities. On randomly sampled leaves, uredinia were counted 9 days after inoculation and urediniospores were collected and quantified every other day from the seventh to the 29th day after inoculation. Increases in inoculum density resulted in progressively smaller increases in uredinial numbers. However, total urediniospore production per leaf was not significantly affected by inoculum, and hence uredinial, density over a range of approximately 10-300 uredinia on the leaf. Total urediniospore production per uredinium generally decreased with increasing inoculum or uredinial density. At high densities, sporulation per uredinium peaked earlier in the sporulation period, had a less distinct peak, and dropped off earlier than for the lower densities. Logistic model fits to cumulative sporulation curves over time revealed that strain SR41 had a greater epidemic rate parameter (r) than SR22 at low and intermediate inoculum or uredinial densities, while SR22 had a higher r-value than SR41 at high density. Both strains also exhibited greater r-values in the presence of the other strain than when alone. Results suggest that strains may have different ecological strategies in their timing of reproduction, and that both intra- and interstrain competition can have complex effects on the temporal dynamics of sporulation in pathogen strains.     

Identification of molecular markers linked to Pm13, an Aegilops longissima gene conferring resistance to powdery mildew in wheat

 RFLP, RAPD, STS and DDRT-PCR techniques were applied to find molecular markers linked to Pm13, an Aegilops longissima gene conferring resistance to powdery mildew in wheat. The experimental strategy was based on the differential comparison of DNAs from common wheat and from common wheat/Ae. longissima recombinant lines carrying short segments of the 3S ^l S chromosome arm containing the Pm13 gene. Sixteen RFLP clones that detect loci previously located in the short arms of group-3 wheat chromosomes were screened for their ability to hybridise to Ae. longissima restriction fragments derived from the 3S ^l S segments introgressed into the recombinant lines. Eight RFLP clones and one STS marker detected 3S ^l S-specific fragments whose location relative to the wheat-alien chromatin breakage point of the recombinant lines was determined. Four amplification products were identified through the screening of about 200 RAPD primers. Their polymorphism was associated with the introgression of the alien DNA. One of the differential fragments was derived from the 3S ^l S DNA segment, while the remaining three corresponded to the replaced 3DS DNA. Further analyses carried out using 40 combinations of DDRT-PCR primers detected an additional reproducible polymorphism associated with the presence of 3S ^l S DNA. In view of their possible utilisation in Pm13 marker-assisted selection, differentially amplified RAPD and DDRT-PCR fragments were cloned, transformed into RFLP markers and converted into STS markers. Received: 23 March 1998 / Accepted: 5 August 1998     

Sequence variation in the CYP51 gene of Blumeria graminis associated with resistance to sterol demethylase inhibiting fungicides

Resistance to sterol 14alpha-demethylase inhibiting fungicides (DMIs) has been correlated with mutations in the CYP51 gene, which encodes the target enzyme eburicol 14alpha-demethylase. To test the hypothesis that variation in the CYP51 gene explains variation for DMI sensitivity in barley and wheat powdery mildew species, this gene was sequenced from isolates of Blumeria graminis f.sp. hordei (Bgh) and f.sp. tritici (Bgt), respectively, which differed in their responses to DMIs in agricultural populations in the UK. Two single-nucleotide mutations in the CYP51 gene, which resulted in the amino acid substitutions Y136F and K147Q, were detected. K147Q is a novel mutation present only in Bgh isolates expressing very high levels of resistance. Sequence analysis of the CYP51 gene from the progeny of a cross between DMI-sensitive and resistant Bgh isolates showed that both mutations segregate with resistance, which is consistent with CYP51 controlling a major portion of DMI resistance. However, genetic analysis of resistance to the DMI triadimenol indicates that mutation of the CYP51 gene is not the only mechanism of resistance operating in B. graminis.     

Changes in lipid composition of Blumeria graminis f.sp. tritici conidia produced on wheat leaves treated with heptanoyl salicylic acid

Treatment of wheat leaves with heptanoyl salicylic acid (HS) and trehalose at concentrations of 0.1 and 15 g l(-1), prior to fungal inoculation, resulted in 40% and 60% protection, respectively, against powdery mildew. The total lipid composition of Blumeria graminis f.sp. tritici (Bgt) conidia, the causal agent of wheat powdery mildew, was compared when produced on wheat leaves, respectively, untreated and treated with the two elicitors, HS and trehalose. An obvious effect was observed on lipid composition (sterol and fatty acid (FA)) of Bgt conidia produced on wheat leaves treated with HS. A total of 16 FA (C12-C24 saturated and unsaturated) as well as unusual methoxylated Fatty Acids (mFA) (3-methoxydocosanoic and 3-methoxytetracosanoic acids) were detected in the conidia. Medium chain FA were predominant in HS treated conidia (64.65%) while long chain fatty acids constituted the major compounds in untreated conidia (62%). The long chain/medium chain FA ratio decreased from 1.8 in the conidia produced on untreated leaves to 0.5 in the conidia obtained from HS treated leaves. When comparing the sterol composition of Bgt conidia produced on leaves treated with HS versus conidia obtained from untreated ones, very important changes within the two major classes can be seen. In particular, 24-methylsterols, e.g., 24-methylenecholesterol and 24-methylcholesta-7,24-dien were reduced by about 82% whereas 24-ethylsterols, e.g., 24-ethylcholesterol and 24-ethylcholesta-5,22-dienol were increased by about 85%. The 24-methylsterols/24-ethylsterols ratio was reduced by ninefold in the conidia produced from HS treated leaves.     

Evolution of resistance against powdery mildew in winter wheat populations conducted under dynamic management. II. Adult plant resistance

The evolution of adult plant resistance towards powdery mildew (caused by Blumeria graminis f. sp. tritici) was investigated in 11 wheat populations cultivated for 10 years in a French network for dynamic management (DM) of wheat genetic resources. The aims of the study were to compare the evolution of resistance in sites submitted to different powdery mildew pressure and to investigate the implication of specific resistance gene action in adult plant resistance. For this, 7 of the 11 populations were characterized for their composition of specific resistance genes (results presented in a former paper). Even though no population differed significantly from the initial PA0 pool for mean adult plant resistance, divergence appeared among the final populations. The populations with the highest adult plant resistance level originated from sites where powdery mildew pressure is known to be high (Vervins, Le Rheu), whereas populations with the lowest adult plant resistance corresponded to areas with no, or very low, powdery mildew pressure (Toulouse, Montreuil-Bellay). A residual effect of defeated specific resistance genes was hypothesized, as lines accumulating at least two specific resistance genes appeared more resistant. Additional quantitative resistance seemed to be involved in adult plant resistance. DM lines appeared then as an interesting source of variability for resistance towards powdery mildew. Moreover, as these lines had been grown in mixed populations they may be appropriate as components of a composite cultivar. Received: 15 December 1999 / Accepted: 30 December 1999     

Melanin in the extracellular matrix of germlings of Botrytis cinerea

Previous work on the composition of the extracellular matrix of germlings of the plant pathogenic fungus Botrytis cinerea demonstrated the presence of carbohydrate, protein, and simple lipids; which, together, comprised 50-60% of the dry weight. Here we show that most of the remaining mass of the extracellular matrix consists of a chemically inert dark pigment with the electron paramagnetic resonance characteristics of a melanin. Scanning electron micrographs of the purified pigment, and transmission electron micrographs of thin sections made using the pigment indicate that it has a filamentous structure. We conclude that melanin is an important component of the extracellular matrix of germlings of B. cinerea. This is the first report of a melanin present in the extracellular matrix of a plant pathogenic fungus.     

Heterologous expression of dammarenediol synthase gene in an engineered Saccharomyces cerevisiae

Aims: Dammarenediol production by an engineered yeast Saccharomyces cerevisiae was investigated. Methods and Results: A dammarenediol‐producing engineered yeast was constructed by heterologous expression of the dammarenediol synthase gene from Panax ginseng hairy roots through RT‐PCR. Fermentation was carried out in a 5‐L GRJY‐bioreactor with an inoculum size of 1% v/v at 30°C. Dammarenediol detection was performed with silica gel chromatography and HPLC. Determination of dammarenediol synthase activity subcellular distribution was carried out by surveying the enzyme activity in microsomes, lipid particles and total yeast homogenate. When cultured under aerobic conditions, the engineered yeast could produce dammarenediol up to 250 μg l^?1. However, when an anaerobic shift strategy was employed, dammarenediol accumulated at a level as twice as that under aerobic condition. The dammarenediol synthase and dammarenediol were mainly localized in lipid particles. Conclusions: Dammarenediol could be heterologously produced in engineered yeast. The heterologously expressed dammarenediol synthase is mainly localized in lipid particles. Anaerobic shift strategy could enhance the dammarenediol level in the engineered yeast. Significance and Impact of the Study: This study showed that the high‐value plant product dammarenediol could be produced by heterologous expression of the according gene in yeast. Furthermore, the anaerobic shift strategy could be potentially applied in oxidosqualene‐derived compounds production in yeast. Here, the information about subcellular distribution of heterologously expressed dammarenediol synthase in the engineered yeast was also provided.     

A barley SKP1‐like protein controls abundance of the susceptibility factor RACB and influences the interaction of barley with the barley powdery mildew fungus

In an increasing number of plant–microbe interactions, it has become evident that the abundance of immunity‐related proteins is controlled by the ubiquitin–26S proteasome system. In the interaction of barley with the biotrophic barley powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh), the RAC/ROP [RAT SARCOMA‐related C3 botulinum toxin substrate/RAT SARCOMA HOMOLOGUE (RHO) of plants] guanosine triphosphatase (GTPase) HvRACB supports the fungus in a compatible interaction. By contrast, barley HvRBK1, a ROP‐binding receptor‐like cytoplasmic kinase that interacts with and can be activated by constitutively activated HvRACB, limits fungal infection success. We have identified a barley type II S‐phase kinase 1‐associated (SKP1)‐like protein (HvSKP1‐like) as a molecular interactor of HvRBK1. SKP1 proteins are subunits of the SKP1‐cullin 1‐F‐box (SCF)–E3 ubiquitin ligase complex that acts in the specific recognition and ubiquitination of protein substrates for subsequent proteasomal degradation. Transient induced gene silencing of either HvSKP1‐like or HvRBK1 increased protein abundance of constitutively activated HvRACB in barley epidermal cells, whereas abundance of dominant negative RACB only weakly increased. In addition, silencing of HvSKP1‐like enhanced the susceptibility of barley to haustorium establishment by Bgh. In summary, our results suggest that HvSKP1‐like, together with HvRBK1, controls the abundance of HvRACB and, at the same time, modulates the outcome of the barley–Bgh interaction. A possible feedback mechanism from RAC/ROP‐activated HvRBK1 on the susceptibility factor HvRACB is discussed.