Leishmania major: expression and gene structure of the glycoprotein 63 molecule in virulent and avirulent clones and strains

Two Leishmania membrane glycoconjugates, gp63 and lipophosphoglycan, have been implicated in parasite attachment and uptake into the host macrophage. Moreover, recent data suggest that parasite virulence is associated with high expression of gp63. In this study we have surveyed gp63 gene copy number, in addition to the level of expression of gp63 mRNA and protein in several Leishmania major isolates, as well as virulent and avirulent strains and clones. The highest level of gp63 expression was found in the avirulent cloned line LRC-L119.3G7, which expresses about a 15-fold higher level of gp63 RNA and protein than the virulent cloned line LRC-L137/7/V121, suggesting that large amounts of gp63 are not sufficient for infectivity and do not correlate with virulence. L119.3G7 has eight copies of the gp63 gene compared to five copies in the virulent cloned line V121 and its parental virulent isolate LRC-L137. A series of avirulent clones derived from LRC-L137 also had five copies of the gene, suggesting that gp63 copy number is maintained among closely related parasites. Different virulent isolates of L. major from different geographic regions exhibited six copies of the gp63 gene. The variation in total gene copy number is due to different numbers of the tandemly repeated gp63 isogene in different strains. Our data show that there is wide variability between strains of L. major in the copy number of gp63 genes as well as in the amount of RNA and protein expressed.     

Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major

Mitchell G. F., Handman E. and Spithill T. W. 1985. Examination of variables in the vaccination of mice against cutaneous leishmaniasis using living avirulent cloned lines and killed promastigotes of Leishmania major. International Journal for Parasitology15: 677–684. In vaccination experiments not involving adjuvants, genetically-susceptible mice were injected with living avirulent cloned promastigotes of Leishmania major or killed promastigotes prior to cutaneous challenge with virulent cloned promastigotes. Emphasis was placed on aspects that may contribute to marked variability between experiments and between laboratories in vaccination of mice against cutaneous leishmaniasis. One variable, the challenge promastigotes, was shown to be important in that cloned virulent parasites (V121) were less pathogenic in terms of rate of cutaneous lesion development, than the parental isolate LRC-L137 when low doses of promastigotes were used and particularly when harvested from the log phase of culture. It is likely that avirulent parasites in mixed isolates can increase the rate of lesion development after cutaneous deposition. As reported previously, intraperitoneal, and more particularly intravenous injections of living avirulent cloned parasites (A12) increase resistance in mice. Most importantly, a difference has been demonstrated in the vaccinating efficacy of killed promastigotes of various isolates injected intravenously. This implies that certain isolates of L. major (e.g. the “Moshkovsky strain”) express “host-protective antigens” at higher levels, or in a qualitatively different manner, than other isolates (e.g. LRC-L137). The finding will greatly facilitate the identification of vaccine antigens in this system using immunochemical and gene cloning approaches.     

Acid phosphatase activity of promastigotes of Leishmania donovani: a marker of virulence

Seven cloned lines of promastigotes of Leishmania donovani (UR 6) were isolated by limiting dilution. One clone, UR6-C25, failed to multiply inside the macrophages of line J774G8 and thus was labelled as avirulent. Another, UR6-C24, multiplied inside macrophages, had a virulence index as high as 93 +/- 9.8 and was thus labelled as highly virulent. The other five clones had variable degree of virulence indices ranging from 46.4 +/- 5.8 to 67.6 +/- 3.5. No significant difference in the degrees of attachment of virulent and avirulent populations of promastigotes to macrophages was observed, suggesting no difference in the ligand utilised by these populations for attachment to the macrophages. Acid phosphatase activity of cloned promastigotes correlated with the degree of virulence. These data suggest that acid phosphatase activity could be used as a marker to differentiate avirulent from virulent populations of promastigotes of L. donovani.     

Passive transfer of Leishmania lipopolysaccharide confers parasite survival in macrophages

Infection of macrophages by the intracellular protozoan parasite Leishmania involves specific attachment to the host membrane, followed by phagocytosis and intracellular survival and growth. Two parasite molecules have been implicated in the attachment event: Leishmania lipopolysaccharide (L-LPS) and a glycoprotein (gp63). This study was designed to clarify the role of L-LPS in infection and the stage in the process of infection at which it operates. We have recently identified a Leishmania major strain (LRC-L119) which lacks the L-LPS molecule and is not infective for hamsters or mice. This parasite was isolated from a gerbil in Kenya and was identified phenotypically as L. major by isoenzyme and fatty acid analysis. In this study we have confirmed at the genotype level that LRC-L119 is L. major by analyzing and comparing the organization of cloned DNA sequences in the genome of different strains of L. major. Here we show that LRC-L119 promastigotes are phagocytosed rapidly by macrophages in vitro, but in contrast to virulent strains of L. major, they are then killed over a period of 18 hr. In addition, we show that transfer of purified L-LPS from a virulent clone of L. major (V121) into LRC-L119 promastigotes confers on them the ability to survive in macrophages in vitro.     

An amphipathic sulphated glycoconjugate of Leishmania: characterization with monoclonal antibodies

A major glycoconjugate of Leishmania tropica major identified by two monoclonal antibodies was shown to be an externally oriented, amphipathic membrane antigen shed into the culture medium in which the parasites grow. This molecule could be labelled metabolically with [3H]glucose, [3H]galactose, [32P]phosphate and [35S]sulphate. It migrated as a polydisperse band upon electrophoresis in SDS-polyacrylamide gels, spanning the region of the gel corresponding to an apparent mol. wt. of 20 000-67 000 daltons. An apparently identical family of molecules could be labelled on the surface of living promastigotes using galactose oxidase and [3H]-sodium borohydride. This molecule was shown to be released into the supernatant over a period of several hours. Detection of the 3H- or 35S-labelled molecule required several days exposure of autoradiographs, but a novel blotting technique using nitrocellulose coated with monoclonal antibody allowed rapid detection of the molecule in charge shift electrophoresis, Western blotting and dot blotting. The electrophoretic mobility of the glycoconjugate in agarose relative to its mobility in Triton X-100 was increased in the presence of deoxycholate, and decreased in the presence of cetyl trimethyl-ammonium bromide, indicating amphipathic properties consistent with insertion into the lipid bilayer of the membrane. Using the dot-blotting technique the glycoconjugate was detected in all virulent and avirulent clones of LRC-L137 and in two additional isolates of L. tropica major (LRC-L287 and LRC-L251), but not in L. donovani or L. mexicana, consistent with the previously described specificity of the antibodies. However, the general approaches used in this paper showed that L. donovani (LRC-L52) and L. mexicana (LRC-L94) synthesize a similar, but antigenically distinct glycoconjugate.     

Leishmania (Leishmania) amazonensis: differential expression of proteinases and cell-surface polypeptides in avirulent and virulent promastigotes

A comparative study of proteolytic enzymes and cell-surface protein composition in virulent and avirulent Leishmania (Leishmania) amazonensis promastigote forms was carried out using one- and two-dimensional dodecyl sulfate sodium-polyacrylamide gel electrophoresis (SDS-PAGE). The surface iodinated protein profiles showed two major polypeptides of 65-60 and 50-47 kDa that were expressed in both virulent and avirulent promastigote forms. However, minor quantitative differences were observed in the cell-surface profile between the avirulent and virulent promastigotes. These included polypeptides of 115, 52, 45, 32, and 25 kDa that were preferentially expressed in the virulent forms. Two-dimensional SDS-PAGE showed an accentuated expression of acidic polypeptides; some of them differentially expressed in the promastigote forms analyzed. Live parasites treated with glycosylphosphatidylinositol (GPI)-specific phospholipase C (PLC) from Trypanosoma brucei and immunoprecipitated with the cross-reacting determinant (CRD) antibody recognized three major polypeptides of 65-60, 52, and 50-47 kDa, hence suggesting that these peptides were anchored to the plasma membrane domains through GPI anchor. Moreover, the polypeptides of 65-60 and 52 kDa were also recognized by the gp63 antiserum. Several metalloproteinase activities were similar in both virulent and avirulent promastigote forms, whereas cysteine proteinase activities, sensitive to E-64, were preferentially expressed in virulent promastigotes. These results suggest that cell-surface polypeptides and intracellular cysteine proteinases might play an important role in the virulence of L. (L.) amazonensis.     

The Leishmania receptor for macrophages is a lipid-containing glycoconjugate.

The glycoconjugate of Leishmania major recognized by the monoclonal antibody WIC-79.3 exists in two forms. The cellular form associated with the promastigote is a population of amphipathic molecules consistent with membrane insertion. In contrast, the extracellular form mainly consists of hydrophilic molecules, and probably arises by cleavage of the cellular form by an endogenous phospholipase. The hydrophilic population of extracellular glycoconjugate molecules binds specifically to macrophages but not to T or B lymphoid cells. Binding of the glycoconjugate and also intact promastigotes to macrophages in vitro is specifically inhibited by Fab fragments of WIC-79.3. These data indicate that the L. major glycoconjugate is the parasite receptor for macrophages, and hence the molecule directly involved in the initiation of infection.     

The generation of infective stage Leishmania major promastigotes is associated with the cell-surface expression and release of a developmentally regulated glycolipid

A monoclonal antibody, 3F12, was generated which reacted specifically against infective or metacyclic stage Leishmania major promastigotes, but not with noninfective promastigotes obtained from log phase cultures. The antibody recognized a cell surface and released molecule that could be metabolically labeled with [14C]glucose, [3H]mannose, [3H]galactose, and [3H]palmitic acid, but not with [35S]methionine or [3H]leucine. The molecule was the major species surface-labeled by [3H]sodium borohydride after periodate treatment. The glycolipid appeared to be shed primarily as free carbohydrate because 70% of the released material partitioned in the aqueous fraction after phase separation in TX-114. The molecule could be distinguished from the L. major glycolipid which has already been extensively described because its migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was of higher relative m.w. However, a close relationship between the two molecules was indicated by the finding that another monoclonal antibody, WIC-79.3, recognized both forms of the glycolipid; one produced and released only by log phase promastigotes, and one produced and released only by metacyclic promastigotes. The loss of agglutination with peanut agglutinin which has been shown to accompany metacyclogenesis was found to be caused by the loss of expression of the log form of the glycolipid which in most cases appeared to be the result of the developmental modification of this molecule. A survey of a number of virulent and avirulent. L. major strains and clones reinforced an absolute association between the ability of these promastigotes to initiate infection in BALB/c mice and their expression and release of the 3F12-binding, developmentally regulated form of the glycolipid. Not only does this glycolipid serve as the first well defined molecular marker for infective stage metacyclic promastigotes, but its unique structure is very likely to contribute to the adaptive changes that allow these parasites to survive within the vertebrate host.     

Virulence attenuation of a UDP-galactose/<Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β1,4 galactosyltransferase expressing <Emphasis Type="Italic">Leishmania donovani</Emphasis> promastigote

Protozoan parasites of the genus Leishmania are the causative agent of leishmaniasis, a disease whose manifestations in humans range from mild cutaneous lesions to fatal visceral infections. Human visceral leishmaniasis is caused by Leishmania donovani. Long-term culture in vitro leads to the attenuation of the parasite. This loss of parasite virulence is associated with the expression of a developmentally regulated UDP-Galactose/N-acetylglucosamine beta 1-4 galactosyltransferase and galactose terminal glycoconjugates as determined by their agglutination with the pea nut agglutinin (PNA). Thus, all promastigotes passaged for more than 11 times were 100% agglutinated with PNA, and represent a homogeneous population of avirulent parasites. Identical concentrations of PNA failed to agglutinate promastigotes passaged for < or =5 times. These PNA(-) promastigotes were virulent. Promastigotes passaged from 5 to 10 times showed a mixed population. The identity of populations defined by virulence and PNA agglutination was confirmed by isolating PNA(+) avirulent and PNA(-) virulent clones from the 7th passage promastigotes. Only the PNA(+) clones triggered macrophage microbicidal activity. The PNA(+) clones lacked lipophosphoglycan. Intravenous administration of [(14)C] galactose-labeled parasite in BALB/c mice resulted in rapid clearance of the parasite from blood with a concomitant accumulation in the liver. By enzymatic assay and RT-PCR we have shown the association of a UDP-Galactose/N-acetylglucosamine beta1,4 galactosyltransferase with only the attenuated clones. By immunofluorescence we demonstrated that the enzyme is located in the Golgi apparatus. By western blot analysis and SDS-PAGE of the affinity-purified protein, we have been able to identify a 29 KDa galactose terminal protein from the avirulent clones.     

Involvement of protein tyrosine kinases and phosphatases in uptake and intracellular replication of virulent and avirulent Leishmania donovani promastigotes in mouse macrophage cells

In this study we show that protein tyrosine kinases (PTKs) and also protein tyrosine phosphatases are involved in the uptake of virulent and avirulent Leishmania donovani promastigotes by macrophage cells. Protein tyrosine kinase inhibitors such as genistein or tyrphostin 25 decrease parasite uptake in a dose-dependent manner. Addition of sodium orthovanadate, a protein tyrosine phosphatase inhibitor, prior to infection significantly increases parasite internalization. A similar uptake profile was observed with both virulent and avirulent L. donovani promastigotes. Treatment of macrophages with cytochalasin B, an inhibitor of actin polymerization prevents promastigote uptake, indicating that a tyrosine kinase induced actin polymerization signal may be necessary for the entry of the parasites. In contrast, neither genistein nor tyrphostin significantly reduce intracellular replication of this pathogen or nitric oxide production, suggesting that the PTK-mediated signal is not related to the ultimate virulence mechanism associated with intracellular replication of this pathogen. These data collectively suggest that protein tyrosine kinase mediated entry of L. donovani promastigotes into macrophages is not a virulence-associated event.     

Overexpression of the Leishmania amazonensis Ca2+-ATPase gene lmaa1 enhances virulence

A gene for a Ca2+-transporting ATPase (lmaa1) from the trypanosomatid parasite Leishmania (mexicana) amazonensis was overexpressed in two clones of L. amazonensis differing in their virulence. RNA and protein expression of the gene was increased in transfectants, as was the infectivity of transfectants versus parental types in both mouse and in vitro macrophage infection experiments. The virulence of the almost avirulent clone was enhanced such that it was more virulent than the parental 'virulent' clone. Growth of the parasites in culture as promastigotes, after isolation from mouse lesions, indicated that transfection led to improved survival of promastigotes during the stationary phase of culture. As it is in this culture phase that infective metacyclic forms develop, the key role of the Lmaa1 protein may be in metacyclogenesis. The protein may be important in the synthesis and trafficking of new proteins through the secretory pathway, as we demonstrate, using a green fluorescent protein hybrid and by immunofluorescence, that the Lmaa1 protein is located in the endoplasmic reticulum in promastigotes and amastigotes of L. amazonensis.     

Differences in human macrophage receptor usage, lysosomal fusion kinetics and survival between logarithmic and metacyclic Leishmania infantum chagasi promastigotes

The obligate intracellular protozoan, Leishmania infantum chagasi (Lic) undergoes receptor-mediated phagocytosis by macrophages followed by a transient delay in phagolysosome maturation. We found differences in the pathway through which virulent Lic metacyclic promastigotes or avirulent logarithmic promastigotes are phagocytosed by human monocyte-derived macrophages (MDMs). Both logarithmic and metacyclic promastigotes entered MDMs through a compartment lined by the third complement receptor (CR3). In contrast, many logarithmic promastigotes entered through vacuoles lined by mannose receptors (MR) whereas most metacyclic promastigotes did not ( P  < 0.005). CR3-positive vacuoles containing metacyclic promastigotes stained for caveolin-1 protein, suggesting CR3 localizes in caveolae during phagocytosis. Following entry, the kinetics of phagolysosomal maturation and intracellular survival also differed. Vacuoles containing metacyclic parasites did not accumulate lysosome-associated membrane protein-1 (LAMP-1) at early times after phagocytosis, whereas vacuoles with logarithmic promastigotes did. MDMs phagocytosed greater numbers of logarithmic than metacyclic promastigotes, yet metacyclics ultimately replicated intracellularly with greater efficiency. These data suggest that virulent metacyclic Leishmania promastigotes fail to ligate macrophage MR, and enter through a path that ultimately enhances intracellular survival. The relatively quiescent entry of virulent Leishmania spp. into macrophages may be accounted for by the ability of metacyclic promastigotes to selectively bypass deleterious entry pathways.     

Modulation of the mitogenic PHA response of carp, Cyprinus carpio L., by extracellular products of Aeromonas salmonicida

The influence of bacteria-free supernatants from cultures of atypical virulent (V234/81, auto-agglutinating. A-layer positive) and avirulent (126/68, non-agglutinating, A-layer negative) strains of A. salmonicida , obtained after different culture times in yeast-tryptone broth at 20°C, was tested on the PHA response of carp pronephric leucocytes in vitro . Supernatants from virulent cultures modulated the response, whereas avirulent supernatants had no effect. The response was enhanced (400%) by supernatant from early virulent cultures (20 h), but severely depressed (<3%) by later ones (96 h). The effects were dose-dependent. Inhibitory activity of 96-h supernatant was lost by heating (70°C, 30 min), suggesting that the inhibiting factors are all proteinaceous.
Heated 96-h supernatant was as stimulatory as early supernatant. Stimulation of leucocytes also occurred in the absence of PHA with early and heat-treated 96-h supernatants, but at a tenth of the level, suggesting that only stimulated cells (blasts) might respond to the substance(s) present in supernatants. Membrane fragments from virulent and avirulent bacteria, and purified LPS from virulent bacteria, were stimulatory with or without PHA. Endotoxin-free, heat-treated, 96-h culture supernatants were also stimulatory, suggesting that an additional mitogenic factor(s), other than LPS, is present in the supernatants. The modulating in vitro effects of extracellular products from A. salmonicida might explain the immunosuppression seen during later stages of erythrodermatitis in vivo.     

Leishmania major: solid phase radioimmunoassay for antibody detection in human cutaneous leishmaniasis

A radioimmunoassay for the quantitative determination of antileishmanial antibody in sera from patients suffering from cutaneous leishmaniasis was developed. The assay, using as antigen either the soluble fraction from freeze-thawed sonicated Leishmania major (LRC-L137) promastigotes or a carbohydrate-lipid containing fraction obtained by extraction with hexane-isopropanol, was shown to be sensitive and reproducible. The sera of 95 patients were examined. These were from patients with cutaneous leishmaniasis (26 from the Jordan Valley and 13 from Sinai), kala-azar (9), malaria (24), schistosomiasis (10), toxoplasmosis (5), and leprosy (8); controls were 37 normal human sera. No significant antigen dependent differences were observed using sera from cutaneous leishmaniasis patients, although differences in the immunological response were observed between the two populations of these patients. Antileishmanial activity was not detected in sera from patients with malaria, schistosomiasis, or toxoplasmosis. Although sera from leprosy patients crossreacted with the carbohydrate-lipid containing fraction, it was nevertheless more strain specific than freeze thawed sonicated L. major.     

Expression of the major surface glycoprotein of Leishmania donovani chagasi in virulent and attenuated promastigotes

Using both hamster and mouse models of infection, we documented that the virulence of Leishmania donovani chagasi promastigotes decreases over time, when parasites are maintained in long term culture after isolation from an infected animal. Concomitant with this loss of virulence is a marked decrease in amount of the major promastigote surface glycoprotein, gp63, present in promastigotes. The latter was shown by a decrease in binding of polyclonal anti-gp63 serum to attenuated (cultivated long term) as compared to virulent (recently isolated) promastigotes, using immunofluorescence and Western blot assays. Binding of Con A to promastigote glycoproteins, separated by SDS-PAGE, documented a similar decrease. An alteration in the mechanism of promastigote attachment to macrophages was also noted: purified gp63 inhibited attachment of virulent promastigotes to human monocyte-derived macrophages, but it did not affect the attachment of attenuated promastigotes. Northern blot analysis showed that, despite marked differences in the amount of gp63 protein, the quantity of gp63 RNA was comparable in attenuated and virulent promastigotes. However, virulent promastigotes contained two major gp63 RNA species of 3.0 and 2.7 kb, whereas attenuated promastigotes had one predominant gp63 RNA of 2.7 kb and only minor amounts of 3.0 kb RNA. Thus, the decrease in gp63 expression in attenuated, contrasted to virulent, promastigotes is associated with qualitative, but not quantitative, differences in the gp63 messenger RNA.     

Isofemale Line Analysis of Meloidogyne incognita Virulence to Cowpea Resistance Gene Rk

Isofemale lines (IFL) from single egg masses were studied for genetic variation in Meloidogyne incognita isolates avirulent and virulent to the resistance gene Rk in cowpea (Vigna unguiculata). In parental isolates cultured on susceptible and resistant cowpea, the virulent isolate contained 100% and the avirulent isolate 7% virulent lineages. Virulence was selected from the avirulent isolate within eight generations on resistant cowpea (lineage selection). In addition, virulence was selected from avirulent females (individual selection). Virulence differed (P ≤ 0.05) both within and between cohorts of IFL cultured for up to 27 generations on susceptible or resistant cowpea. Distinct virulence profiles were observed among IFL. Some remained avirulent on susceptible plants and became extinct on resistant plants; some remained virulent on resistant and susceptible plants; some changed from avirulent to virulent on resistant plants; and others changed from virulent to avirulent on susceptible plants. Also, some IFL increased in virulence on susceptible plants. Single descent lines from IFL showed similar patterns of virulence for up to six generations. These results revealed considerable genetic variation in virulence in a mitotic parthenogenetic nematode population. The frequencies of lineages with stable or changeable virulence and avirulence phenotypes determined the overall virulence potential of the population.     

Heat-stress induced modulation of protein phosphorylation in virulent promastigotes of Leishmania donovani

In parasites such as Leishmania, the study of molecular events induced in response to heat stress is of immense interest since temperature increase is an integral part of the life cycle. Protein phosphorylation is known to control major steps of proliferation and differentiation in eukaryotic cells. Studies on intracellular signaling systems in protozoa are relatively recent. We have examined the effect of heat shock on the protein phosphorylation status in promastigotes of Leishmania donovani. The patterns of total protein phosphorylation and specific phosphorylation at tyrosine residues were examined using [32P]-orthophosphate labelling of the parasites and immunoblotting with a monoclonal anti-phosphotyrosine antibody. The major proteins of L. donovani that were phosphorylated at 24 degrees C had apparent molecular weights of 110, 105, 66-68, 55, 36-40 and 20 kDa. Heat shock (from 24 to 37 degrees C) led to a significant decrease in phosphorylation of the majority of phosphoproteins in the virulent promastigotes. On the other hand, the avirulent promastigotes did not show any decrease in protein phosphorylation on exposure to heat stress. Predominant phosphorylation at tyrosine residues was detectable in proteins of putative size 105-110 kDa in both virulent and avirulent parasites. Heat shock led to a reduction in the level of phosphotyrosine in both these proteins in the case of virulent parasites, while no such reduction was detectable in avirulent parasites. Significant modifications in the phosphorylation status of proteins in response to heat stress including that of tyrosine containing proteins, observed exclusively in virulent parasites, suggest that modulation of protein phosphorylation/dephosphorylation may play a role in signal transduction pathways in the parasite upon heat shock encountered on entering the mammalian host.     

Changing surface carbohydrate configurations during the growth of Leishmania major.

Surface carbohydrates of leishmanial promastigotes change during their growth cycle. These changes were monitored in a cloned line of Leishmania major in 2 media. A freshly isolated virulent strain was also examined. Infectivity, surface sugar moieties, and released glycoconjugates (EF) were examined and compared during the growth cycle. When promastigotes of the same clone were grown in different media, their lectin-mediated agglutination profiles were dissimilar, and both quantitative and qualitative variation was seen in the antigenic glycoconjugates released into the media. When labeled with fluorescent lectins, the virulent strain showed no loss of galactose, an increase of N-acetylglucosamine, and a midcycle decrease of N-acetylgalactosamine. Promastigotes of this strain were infective to hamsters throughout the growth cycle. The results presented indicate that culture medium components regulate carbohydrate surface configurations and, thereby, antigenic expression. Infectivity of the virulent strain was not dependent on the expression of a single surface carbohydrate.     

爪哇根结线虫Mi-毒性和无毒近等基因系的制备及其遗传变异的初步分析

爪哇根结线虫是以孤雌生殖方式繁殖的农作物重要病原物,通过将爪哇根结线虫-无毒群体在抗病番茄品种Momotaro上连续选择19代,获得了一对针对抗性基因Mi的毒性和无毒近等基因系,用102种引物对该近等基因系进行随机扩增多态性DNA（RAPD）分析显示,除一种引物外两者的RAPD带谱基本一致,证实了该毒性和无毒群体的基因组组成十分近似,对一种引物扩增出的一个无毒群体特异性的RAPD片段进行了克隆,Southern杂交结果提示该多态性片段及其同源序列在毒性选择过程中被从毒性线虫的基因组中删除,但DNA序列分析和数据库检索表明,该片段与迄今发表的核酸序列均不存在显著的同源性,因此尚无法预测其潜在的功能性,爪哇根结线虫Mi-毒性和无毒近等基因系的制备为进一步鉴定和分离该线虫毒性相关基因打下了坚实的基础。     

Screening for Spontaneous Virulent Mutants of Erysiphe graminis D.C. f.sp. hordei on Barley lines with Resistance Genes Ml-a1, Ml-a6, Ml-a12 and Ml-g

Seedlings of 4 barley lines with powdery mildew resistance genes Ml-a1, Ml-a6 Ml-a12 and Ml-g were inoculated with powdery mildew culture CR3 which is avirulent to the 4 host lines. The inoculation density was 1.2 infectious conidia per mm², and in total 50 million conidia were screened for the occurrence of virulent mutans. During 30 cycles of screening, 43 putative virulent mutants were selected, multiplied and tested. They could be grouped in 5 different genotypes according to virulence spectrum. Based on the virulence spectre, mating type, biochemical tests and analyses of test crosses, 3 of the types were rejected as being of mutational origin, and the verification of the remaining 2 were not consistent with the expectations deduced from a gene-for-gene interaction. Provided that none of the genotypes found were of mutational origin, the spontaneous mutation frequency from avirulence to virulence in barley powdery mildew is therefore below 2 × 10^–8. A reconstructation experiment showed that the density of avirulent inoculum did not reduce the survival rate of rate virulent genotypes