2012 - Washington Red Raspberry Commission

We will collect large quantities of P. penetrans and clean the nematodes from contaminating
plant material and other nematode species. Root-lesion nematodes will be stored in an ultra-low
freezer at -80 ºC. Once a sufficient amount of nematode material has been obtained, we will
isolate mRNA, which represents the message of genes. To create a collection of all P. penetrans
genes, we will sequence all genes using high-throughput sequencing technologies. Once
sequence data has been obtained, we will process it to remove contaminating sequences from
plants and other organisms. The more active a given gene is, the more sequences we will obtain
for that particular gene. We will sort all sequence data and generate a profile for all genes that
will show us a range from the most to the least active nematode genes.
2) Identify P. penetrans secretion genes and effector gene candidates.
To separate P. penetrans genes that produce secretions from other genes, we will search for
genes that contain a signal peptide-coding region using SignalP software (Bendtsen et al. 2004).
The signal peptide region is a hallmark of genes that produce secretions and is therefore present
in all effector genes. Once we have narrowed down the total gene library to those genes that
produce secretions, we will screen for the most active effector genes. To this end, we will use a
technique, in situ hybridization, which employs a color reaction specific to individual gene
sequences in question. By using a microscope, we will be able to determine whether the
produced pigment is present in P. penetrans salivary gland cells. This would indicate that the
respective gene is active at that location and therefore a potential effector gene (de Boer et al.
1998). We will focus on those effector gene candidates that have the most sequence data and are
therefore most active, which will facilitate further screening steps. Simultaneously, we will
compare P. penetrans genes with genes in other root-lesion nematode species to find putative
effectors. We anticipate that addressing objective 2 will take up the majority of this funding year.
Anticipated Benefits and Information Transfer:
The identification of P. penetrans effector genes is important, because once their identities are
known, effector genes can be used as control targets. Blocking their functions would result in
increased P. penetrans resistance. The research proposed here is important and benefits
Washington’s raspberry growers, because it aids in developing new nematode control methods
that reduce the use of costly nematicides and that provide an alternative control tactic as part of
an integrated pest management program. Results will be disseminated through presentations at
grower meetings and scientific congresses, and through publications in trade magazines and
scientific journals.
References:
Bendtsen, J.D., Nielsen, H., von Heijne, G. and Brunak, S. 2004. Improved prediction of signal
peptides: SignalP 3.0. Journal of Molecular Biology 340: 783-795.
de Boer, J.M., Yan, Y., Smant, G., Davis, E.L. and Baum, T.J. 1998. In-situ hybridization to
messenger RNA in Heterodera glycines. Journal of Nematology 30: 309-312.
Gao, B., Allen, R., Maier, T., Davis, E.L., Baum, T.J. and Hussey, R.S. 2003. The parasitome of
the phytonematode Heterodera glycines. Molecular Plant-Microbe Interactions 16: 720-726.
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