The parasitome of the phytonematode Heterodera glycines

Parasitism genes expressed in the esophageal gland cells of phytonematodes encode secretions that control the complex process of plant parasitism. In the soybean cyst nematode, Heterodera glycines, the parasitome, i.e., the secreted products of parasitism genes, facilitate nematode migration in soybean roots and mediate the modification of root cells into elaborate feeding cells required to support the growth and development of the nematode. With very few exceptions, the identities of these secretions are unknown, and the mechanisms of cyst nematode parasitism, therefore, remain obscure. The most direct and efficient approach for cloning parasitism genes and rapidly advancing our understanding of the molecular interactions during nematode parasitism of plants is to create gland cell-specific cDNA libraries using cytoplasm microaspirated from the esophageal gland cells of various parasitic stages. By combining expressed sequence tag analysis of a gland cell cDNA library with high throughput in situ expression localization of clones encoding secretory proteins, we obtained the first comprehensive parasitome profile for a parasitic nematode. We identified 51 new H. glycines gland-expressed candidate parasitism genes, of which 38 genes constitute completely novel sequences. Individual parasitome members showed distinct gland cell expression patterns throughout the parasitic cycle. The parasitome complexity discovered paints a more elaborate picture of host cellular events under specific control by the nematode parasite than previously hypothesized.     

In planta secretion of a calreticulin by migratory and sedentary stages of root-knot nematode

Esophageal secretions from endoparasitic sedentary nematodes are thought to play key roles throughout plant parasitism, in particular during the invasion of the root tissue and the initiation and maintenance of the nematode feeding site (NFS) essential for nematode development. The secretion in planta of esophageal cell-wall-degrading enzymes by migratory juveniles has been shown, suggesting a role for these enzymes in the invasion phase. Nevertheless, the secretion of an esophageal gland protein into the NFS by nematode sedentary stages has never been demonstrated. The calreticulin Mi-CRT is a protein synthesized in the esophageal glands of the root-knot nematode Meloidogyne incognita. After three-dimensional modeling of the Mi-CRT protein, a surface peptide was selected to raise specific antibodies. In planta immunolocalization showed that Mi-CRT is secreted by migratory and sedentary stage nematodes, suggesting a role for Mi-CRT throughout parasitism. During the maintenance of the NFS, the secreted Mi-CRT was localized outside the nematode at the tip of the stylet. In addition, Mi-CRT accumulation was observed along the cell wall of the giant cells that compose the feeding site, providing evidence for a nematode esophageal protein secretion into the NFS.     

Immunoproteomics of extracellular proteins of Chinese virulent strains of Streptococcus suis type 2

Streptococcus suis type 2 (SS2) is a porcine zoonotic pathogen with worldwide distribution, and lacking suitable vaccine and virulent maker were bottleneck to control this infection. An immunoproteomic assay was used to identify antigenic proteins from the total extracellular proteins of the virulent Chinese SS2 strain ZY05719. The convalescent serum of a specific pathogen free (SPF) mini-pig recognized nine protein spots on PVDF membrane. Antigenic proteins on a duplicate gel, as well as those with a similar placement of extracellular proteins from another virulent strain (HA9801) and an avirulent strain (T15) on 2-D gels, were excised and identified by MALDI-TOF-MS. PMF of the protein spots were performed using the MASCOT server. Two proteins were found in all three strains. Comparative proteomic analysis between the two virulent strains and the avirulent strain revealed nine differential proteins, eight of which were successfully identified. Genes for six of the differentially expressed proteins were found in both virulent strains, and of those were present in the avirulent stain.     

Differential accumulation of poly(A)+ RNA between virulent and double-stranded RNA-induced hypovirulent strains of Cryphonectria (Endothia) parasitica.

The double-stranded RNA responsible for transmissible hypovirulence in Cryphonectria (Endothia) parasitica was found to affect the accumulation of specific poly(A)+ RNA. Using differential hybridization techniques, two genes were isolated, Vir1 and Vir2, which were specifically expressed as poly(A)+ RNAs in the virulent cells. The highly expressed RNA sequences from these genes were not found in total RNA isolated from either American or European hypovirulent strains, although the genes were present in their genomes. Other virulence- and hypovirulence-specific RNA sequences were also detected. One isolated hypovirulence-specific RNA sequence was expressed in both virulent and hypovirulent cells, but in a two- to fourfold-higher concentration in the hypovirulent cells. The results show that hypovirulence is associated with concurrent changes in a few highly expressed poly(A)+ RNAs, which suggests a specific effect of the double-stranded RNA on fungal gene expression.     

Monoclonal Antibodies to the Esophageal Glands and Stylet Secretions of Heterodera glycines

Three monodonal antibodies (MAbs) that bound to secretory granules within the subventral esophageal glands of second-stage juveniles (J2) of the soybean cyst nematode (SCN), Heterodera glycines, were developed from intrasplenic immunizations of a mouse with homogenates of SCN J2. Two MAbs to the secretory granules within subventral glands and one MAb to granules within the dorsal esophageal gland of SCN J2 were developed by intrasplenic immunizations with J2 stylet secretions. Stylet secretions, produced in vitro by incubating SCN J2 in 5-methoxy DMT oxalate, were solubilized with a high pH buffer and concentrated for use as antigen. Three of the five MAbs specific to the subventral esophageal glands bound to stylet secretions from SCN J2 in immunofluorescence and ELISA assays. Two of these three MAbs also bound to secretory granules within both the dorsal and subventral esophageal glands of young SCN females. All five of the subventral gland MAbs bound to the subventral glands of Heterodera schachtii and one bound to the subventral glands of Globodera tabacum, but none bound to any structures in Meloidogyne incognita or Caenorhabditis elegans.     

Virulence conversion of Legionella pneumophila by conjugal transfer of chromosomal DNA

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.     

Virulence of Meloidogyne incognita to expression of N gene in pepper

Four pepper genotypes classified as resistant and four pepper genotypes classified as susceptible to several avirulent populations of M. incognita were compared for their reactions against a population of Meloidogyne incognita (Chitwood) Kofoid and White which had been shown to be virulent to resistant bell pepper (Capsicum annuum) in preliminary tests. The virulent population of M. incognita originated from a commercial bell pepper field in California. The resistant pepper genotypes used in all experiments were the Capsicum annuum cultivars Charleston Belle, Carolina Wonder, and Carolina Cayenne, and the C. chinense cultigen PA-426. The susceptible pepper genotypes used in the experiments were the C. annuum cultivars Keystone Resistant Giant, Yolo Wonder B, California Wonder, and the C. chinense cultigen PA-350. Root gall indices (GI) were ≥ 3.0 for all genotypes in both tests except for PA-426 (GI=2.57) in test 1 and 'Carolina Cayenne' (GI=2.83) in test 2. Numbers of eggs per gram fresh root weight ranged from 20,635 to 141,319 and reproductive indices ranged from 1.20 to 27.2 for the pepper genotypes in both tests, indicating that all eight pepper genotypes tested were susceptible to the M. incognita population used in these tests. The M. incognita population used in these studies overcame resistance conferred by the N gene in all resistant genotypes of both C. annuum and C. chinense.     

Cloning and characterization of an esophageal-gland-specific chorismate mutase from the phytoparasitic nematode Meloidogyne javanica

Root-knot nematodes are obligate plant parasites that alter plant cell growth and development by inducing the formation of giant feeder cells. It is thought that nematodes inject secretions from their esophageal glands into plant cells while feeding, and that these secretions cause giant cell formation. To elucidate the mechanisms underlying the formation of giant cells, a strategy was developed to clone esophageal gland genes from the root-knot nematode Meloidogyne javanica. One clone, shown to be expressed in the nematode's esophageal gland, codes for a potentially secreted chorismate mutase (CM). CM is a key branch-point regulatory enzyme in the shikimate pathway and converts chorismate to prephenate, a precursor of phenylalanine and tyrosine. The shikimate pathway is not found in animals, but in plants, where it produces aromatic amino acids and derivative compounds that play critical roles in growth and defense. Therefore, we hypothesize that this CM is involved in allowing nematodes to parasitize plants.     

Genome-wide transcriptome analysis of porcine epidemic diarrhea virus virulent or avirulent strain-infected porcine small intestinal epithelial cells

Porcine epidemic diarrhea virus (PEDV) is the main cause of diarrhea, vomiting, and mortality in pigs, which results in devastating economic loss to the pig industry around the globe. In recent years, the advent of RNA-sequencing technologies has led to delineate host responses at late stages of PEDV infection; however, the comparative analysis of host responses to early-stage infection of virulent and avirulent PEDV strains is currently unknown. Here, using the BGI DNBSEQ RNA-sequencing, we performed global gene expression profiles of pig intestinal epithelial cells infected with virulent (GDS01) or avirulent (HX) PEDV strains for 3, 6, and 12 h. It was observed that over half of all significantly dysregulated genes in both infection groups exhibited a down-regulated expression pattern. Functional enrichment analyses indicated that the differentially expressed genes (DEGs) in the GDS01 group were predominantly related to autophagy and apoptosis, whereas the genes showing the differential expression in the HX group were strongly enriched in immune responses/inflammation. Among the DEGs, the functional association of TLR3 and IFIT2 genes with the HX and GDS01 strains replication was experimentally validated by TLR3 inhibition and IFIT2 overexpression systems in cultured cells. TLR3 expression was found to inhibit HX strain, but not GDS01 strain, replication by enhancing the IFIT2 expression in infected cells. In conclusion, our study highlights similarities and differences in gene expression patterns and cellular processes/pathways altered at the early-stage infection of PEDV virulent and avirulent strains. These findings may provide a foundation for establishing novel therapies to control PEDV infection.     

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.     

A gene from Xanthomonas campestris pv. vesicatoria that determines avirulence in tomato is related to avrBs3.

Strains of Xanthomonas campestris pv. vesicatoria that were avirulent in tomato leaves but virulent in pepper leaves were identified. A cloned gene, avrBsP, from one of the strains, Xv 87-7, converted a virulent strain in tomato to avirulent in tomato. A 1.7-kb subclone containing the avirulence gene cross-hybridized with the avirulence gene, which determines race 1 within the pepper group of strains (avrBs3). However, the two avirulence genes differ in their biological activity. The base sequences of the two avirulence genes were almost identical through the 1.7-kb segment of avrBsP, with significant differences only in some bases in the repeat region.     

Identification of putative parasitism genes expressed in the esophageal gland cells of the soybean cyst nematode Heterodera glycines

Cloning parasitism genes encoding secretory proteins expressed in the esophageal gland cells is the key to understanding the molecular basis of nematode parasitism of plants. Suppression subtractive hybridization (SSH) with the microaspirated contents from Heterodera glycines esophageal gland cells and intestinal region was used to isolate genes expressed preferentially in the gland cells of parasitic stages. Twenty-three unique cDNA sequences from a SSH cDNA library were identified and hybridized to the genomic DNA of H. glycines in Southern blots. Full-length cDNAs of 21 clones were obtained by screening a gland-cell long-distance polymerase chain reaction cDNA library. Deduced proteins of 10 clones were preceded by a signal peptide for secretion, and PSORT II computer analysis predicted eight proteins as extracellular, one as nuclear, and one as plasmalemma localized. In situ hybridization showed that four of the predicted extracellular clones were expressed specifically in the dorsal gland cell, one in the subventral gland cells, and three in the intestine in H. glycines. The predicted nuclear clone and the plasmalemma-localized clone were expressed in the subventral gland cells and the dorsal gland cell, respectively. SSH is an efficient method for cloning putative parasitism genes encoding esophageal gland cell secretory proteins that may have a role in H. glycines parasitism of soybean.     

Hsp70 protects macrophages infected with Salmonella choleraesuis against TNF-α-induced cell death

Hsp70 plays an important role in cytoprotection against tumor necrosis factor (TNF) α-mediated cytotoxicity. To investigate the role of Hsp70 in cytoprotein during Salmonella infection, we examined endogenous Hsp70 induction and TNF-α production in a monocyte/macrophage line, J774A.1, after infection with a virulent strain of Salm.choleraesuis RF-1 carrying a 50 kb virulent plasmid or the plasmid-cured avirulent strain 31N-1. Intracellular bacteria progressively increased in J774A.1 cells phagocytosing avirulent 31N-1 bacteria, whereas such progressive growth was not evident in J774A.1 cells phagocytosing avirulent 31N-1 bacteria. On the contrary, J774A.1 cells infected with virulent RF-1 bacteria expressed less Hsp70 than those infected with avirulent 31N-1 bacteria. The level of TNF-α production by J774A.1 infected with virulent RF-1 was much the same as that by J774A.1 infected with avirulent 31N-1. J774A.1 infected with virulent RF-1 died spontaneously; death was inhibited by the addition of anti-TNF-α mAb. Although the frequency of dead J774A.1 with hypodiploid DNA content increased only marginally after infection with avirulent 31N-1, treatment with Hsp70 anti-sense oligonucleotide resulted in a dramatic increase of dead cells in the infected macrophages. Taken together, these results suggest that Hsp70 induced macrophages plays an important role in host defense against Salmonella infection by protecting the macrophages against TNF α-induced cell death. Furthermore, cell death due to impaired endogenous Hsp synthesis in the phagocytes implies a novel pathogenic mechanism for virulence of Salm. choleraesuis RF-1.     

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。     

Cloning and characterization of pectate lyases expressed in the esophageal gland of the pine wood nematode Bursaphelenchus xylophilus

Two pectate lyase genes (Bx-pel-1 and Bx-pel-2) were cloned from the pine wood nematode, Bursaphelenchus xylophilus. The deduced amino acid sequences of these pectate lyases are most similar to polysaccharide lyase family 3 proteins. Recombinant BxPEL1 showed highest activity on polygalacturonic acid and lower activity on more highly methylated pectin. Recombinant BxPEL1 demonstrated full dependency on Ca2+ for activity and optimal activity at 55 degrees C and pH 8 to 10 like other pectate lyases of polysaccharide lyase family 3. The protein sequences have predicted signal peptides at their N-termini and the genes are expressed solely in the esophageal gland cells of the nematode, indicating that the pectate lyases could be secreted into plant tissues to help feeding and migration in the tree. This study suggests that pectate lyases are widely distributed in plant-parasitic nematodes and play an important role in plant-nematode interactions.