[
International Worm Meeting,
2005]
Every year, plant parasitic nematodes cause over $100 billion in damages due to crop loss and plant damage.<span style="mso-spacerun: yes"> </span>Chemical pesticides are the most widespread form of PPN control, but with the prohibition of methyl bromide other approaches to control are necessary.<span style="mso-spacerun: yes"> </span>One approach under consideration is <span style='font-family:"Times New Roman"'>Bacillus thuringiensis</span><span style='font-family:"Times New Roman"'> </span>Bt<span style='font-style: normal'> Crystal (Cry) proteins. Crystal proteins produced by </span>Bt<span style='font-style:normal'> </span><span style='font-family:"Times New Roman"'>have been commonly used for biocontrol of insects.<span style="mso-spacerun: yes"> </span>Our lab has demonstrated that some Bt</span><span style='font-family:"Times New Roman"'> Cry proteins kill free-living nematodes and the free-living stages of one animal parasitic nematode (Nippostrongylus brasiliensis</span><span style='font-family:"Times New Roman"'>).<span style="mso-spacerun: yes"> </span>These results then set the stage for testing whether Cry proteins can control plant-parasitic nematodes.Since most</span> plant parasitic nematodes are obligate endoparasites that feed and develop inside the plant root, delivering <span style='font-family:"Times New Roman"'>Bt</span> Cry protein requires the proteins be expressed directly in the plant. The Cry proteins would then transferred to the nematode through feeding.Our goal is to establish a reliable system for testing transgenic roots for control of endoparasitic nematodes.<span style="mso-spacerun: yes"> </span>This involves expressing the proteins in an appropriate plant and/or root system, infecting those roots with an appropriate nematode host, and then quantitating various parameters of infection.<span style="mso-spacerun: yes"> </span><span style='font-family:"Times New Roman"'>We have synthesized plant-friendly versions of two of these toxins, Cry 6A and Cry 5B.<span style="mso-spacerun: yes"> </span>These were transformed in tomato roots using Agrobacterum rhizogenes</span><span style='font-family:"Times New Roman"'>(hairy roots) and into Arabidopsis using A. tumefaciens</span><span style='font-family:"Times New Roman"'>.<span style="mso-spacerun: yes"> </span>These plant systems are both candidates for infection by root-knot nematode, Meloidogyne incognita.We tested different growth media, lighting conditions, and inoculum levels, in order to optimize number of infections and to develop a reliable system to test the effect of Cry toxins on M. incognita</span><span style='font-family: "Times New Roman"'>.<span style="mso-spacerun: yes"> </span>The experimental conditions and results will be presented.
[
International Worm Meeting,
2005]
Pathogenic bacteria can produce a wide range of gene products that contribute to their virulence in a variety of ways. Bacillus thuringiensis (Bt) is an invertebrate-specific pathogen that produces a family of pore-forming virulence factors with insecticidal or nematicidal activities. These toxins, known as Cry toxins, work by binding to receptors on the surface of the hosts intestine and inserting into the bilayer to form a pore in the host cell that enables the passage of ions and slowly leads to death of the animal. Although pore-formation is common to many bacterial toxins, how it contributes to bacterial virulence and how and to what effect host cells respond to this kind of attack are often unclear.Caenorhabditis elegans is susceptible to the nematicidal Bt Cry toxin Cry5B. Our lab has characterized the genes involved in host response and defense to this toxin. We have used Affymetrix gene chips to study the effect of Cry5B on gene activity. The results show that the animals respond rapidly and robustly at the level of gene expression. Further study of these toxin-responsive genes led to the discovery that two signal transduction pathways function in host defense to Cry toxins. For at least one these pathways, this defensive role is conserved in mammalian cell systems exposed to a vertebrate-specific pore-forming toxin. That pathway is the PMK-1/p38-type mitogen-activated protein (MAP) kinase cascade, a pathway that also mediates worm immunity to bacterial pathogens.We further investigated the role of this pathway in Cry5B defense using microarray analysis to identify the toxin-indcued transcriptional targets of the pathway. An RNAi-based screen of these target genes led to the identification of the ttm gene class (toxin-regulated target of MAP kinase). These genes mediate defense to Cry5B and are transcriptionally activated by Cry5B via the PMK-1 pathway. A description of these genes and possible models for their defensive mechanisms will be presented.We are interested in how pore-forming toxins activate the
p38 pathway. As such, we are measuring levels of phosphorylated PMK-1 in C. elegans exposed to Cry5B to determine if the toxin can serve as a signal for induction of the MAP kinase cascade. Further genetic studies will be aimed at exploring the mechanism of this induction and its significance in the defense response.
[
International Worm Meeting,
2007]
The nematode genus Strongyloides consists of parasites that live as parthenogenetic females in the small intestines of their hosts. In addition to producing parasitic offspring, they can also form a facultative free-living generation with males and females. In most Strongyloides species the progeny of these free-living adults is uniformously female and infective. Based on cytological observations in multiple species several authors1,2,3 have proposed that males do not contribute genetically to the progeny of the free-living generation but that the sperm is merely required to induce parthenogenetic development of the oocyte (pseudogamy). In contrast to these findings, Viney and colleagues4 have found that genetic markers can be passed on to the next generation in S. ratti. We are analyzing the mode of reproduction of the free-living generation of S. papillosus, a parasite of ruminants, cytologically and genetically. We have shown that also in S. papillosus males do contribute genetic material to the next generation5. While some genetic markers are inherited in a manner that is consistent with standard, mendelian, autosomal inheritance, others behave differently in that heterozygous males pass on preferentially or exclusively only one of their two alleles. We are currently testing the hypothesis that this is the consequence of the particular mode of sex determination in S. papillosus. In contrast to most other Strongyloides species that employ an XX/XO sex determining system, in S. papillosus males have a chromosome pair where one of the homologous chromosomes is intact and the other one lacks a large portion as the result of a sex specific chromatin diminution event6. In the process of our work we have noticed that the taxon S. papillosus, most probably, does not reflect a true biological species, but comprises of at least two relatively closely related but reproductively isolated species that can occur as mixed infections in the same host individual. We are currently analyzing the distribution of these two species in local sheep and cattle populations. 1Nigon, V., Roman, E., 1952 Bull biol Fr Belg 86:404-448. 2Bolla, R.I., Roberts, L.S., 1968 J Parasitol 54:849-855. 3Triantaphyllou, A.C., Moncol, D.J., 1977 J Parasitol 63:961-973. 4Viney, M.E., Matthews, B.E., Walliker, D., 1993 Proc R Soc Lond B Biol Sci 254:213-219. 5Eberhardt, A.G., Mayer, W.E., Streit, A., 2007 Int J Parasitol., in press. 6Albertson, D.G., Nwaorgu, O.C., Sulston, J.E., 1979 Chromosoma 75, 75-87.
[
Worm Breeder's Gazette,
1995]
Collagen is the major structural component of nematode cuticles. In Caenorhabditis elegans, the N-terminal regions of cuticle collagen proteins contain the highly conserved motif, R-X-X-R, which has been predicted to contain a potential subtilisin-like proteolytic processing site required for cleavage of the collagen proteins and normal cuticle collagen function (Kramer, 1994, FASEB 8:329-336). Cuticles of adult females of the plant-parasitic nematode Meloidogyne incognita contain a 76 kDa collagen which comprises over 50% of the B- mercaptoethanol-soluble cuticular proteins (Reddigari et al., 1986, J. Nematol. 18:294-302). We determined the N-terminal amino acid sequence of this major collagen and found it to be identical to the predicted amino acid sequence, starting at amino acid number 66, of the M. incognita Lemmi 5 cDNA clone (Van der Eycken et al., 1994, Gene 151:237-242) (Fig.1). A putative subtilisin-like protease recognition site was found immediately upstream of the region of amino acid homology between LEMMI 5 and the N-terminal sequence of the 76 kDa collagen (Fig.1). Our data support previous speculation about the existence of this novel method of collagen maturation and provide further evidence that this mechanism has been conserved during nematode evolution. In addition to protein processing, the expression of the Lemmi 5 gene was transcriptionally regulated: Lemmi 5-specific transcripts were present in adult females but not in eggs or second-stage juveniles. Also, Lemmi 5 analogs were present only in four Meloidogyne species, but not in C. elegans, Heterodera glycines, or tomato. .. PREDICTED LEMMI5 AMINO ACID SEQUENCE .. 1M A T L V V M P Q L Y S Q I N D L N L R V R D G V Q A .. F R V N T D S A W N D L M E L Q V A V T P Q S K P R S * N P F Q S L YR Q K RS L P D Y C I C Q P L E I N79 S L P D Y C I C Q P L E I N ............................................................ CONFIRMED N-TERMINAL AMINO ACID SEQUENCE .............................................................. Figure 1. The partial predicted amino acid sequence (residues 1-79) of the Lemmi 5 collagen gene is shown in roman letters. The putative subtilisin-like protease recognition sequence, R-Q-K-R, is boxed and the first amino acid after the proteolytic cleavage site is indicated by an *. The empirically determined N-terminal sequence of the 76 kDa from adult female M. incognita is italicized.