Abstract

There has been tremendous advancement in analysis and identification of the functional nematode effector genes. At the same time, research has been emphasized on cloning and characterization of the nematode parasitism genes of effector proteins. These are normally secreted through the stylet of the nematode that promotes nematode parasitism in plants. To date, cloning has been achieved for at least 100 parasitism genes while host functions and targets for the secreted effector genes have been elucidated. Therefore, the current research will seek to identify the gene sequences for the effector depending on profile expression, the prediction of signal transmembrane/peptide domain and comparison with the other nematode species. The identified genes will be cloned using qPCR and experiments will be used to identify the spatial expression patterns using in situ hybridisation process.
The plant-parasitic nematodes form the biotrophic parasites that feed from cytoplasm of the unmodified living cells of a plant. The nematodes modify the cells into discrete feeding cells to enable them obtain nutrients that are necessary for growth and development. The current project will identify the effector genes sequences depending on the expression profiles and the identified genes will be cloned using qPCR while the expression will be identified using in situ hybridisation. The project will help in determining the expression for putative effector genes. This will help in generating a transcriptome assembly for the nematode to conform to the silico gene expressions.
Figure 1.0: Schematic representation of phylum nematode with examples of nematode species in brackets and parasite species shown for each branch
Source (Holterman et al. (2006) and Blaxter et al. (1998))
Problem Statement
The evolution of plant parasitic nematodes can be traced back to the biotrophic interactions with the host. The secretion from nematode gland penetrates the root cells through the stylet resulting into profound changes within the gene expression of the plant and the cell structure. The transcriptomes have recently been sequenced for three plant parasitic nematodes.
Aims and objectives of the research
i. To assign an effector function to the gene
ii. Cloning of effector genes
iii. To characterise the nematode effector genes
iv. To compare effector gene complement for Rotylenchulusreniformis and potato cyst nematode
Background to the research
Over the last one decade, there has been extensive research on parasitic nematode effector genes. Such surveys have triggered discussions on pathogens and their economic effects. The plant parasitic nematodes portray a wide variety of the parasite-host interactions. All of them contain a stylet that penetrates the cells to allow feeding. Some of the nematodes are migratory ectoparasites while others are migratory endoparasites. Nevertheless, cyst and root-knot nematodes are the most economically important and are biotrophic in nature. There are over 4100 parasitic nematodes but this research will focus on Rotylrnchulusreniformisand potato cyst nematode (Aires & Rosa 2009, p. 327).
Potato Cyst Nematodes (Heterodera and Globodera Spp.)
Cyst nematodes form the obligate biotrophs that have great economic importance worldwide. The soybean cyst nematodes (SCNs) are the most damaging species among others such as those for potatoes and cereals. The accounting of economic losses has been difficult though for SCNs, the estimated losses are $US1.5billion annually in UK alone(Aires & Rosa 2009, p. 328). The major problem is that the nematodes can survive in soil for prolonged periods and this makes eradicationdifficult (Bauters & Mohammad 2013, p. 379).
Lifecycle
Cyst nematodes moult into an egg in J2 stage. This stage is dormant and is host-specific. Hatching occurs based on the host-derived chemical cues that are present in the root diffusates (Perry, 2002). J2 locates its host by invading and migrating intracellularly and destructively through the root into the inner cortex where the behaviour changes. J2 inserts the stylet into the root cells and waits for the response of the cell. The stylet retracts when it is covered with cellulose layer or after bursting of the protoplasm. This occurs repeatedly until it reaches a cell that is not adversely affected by J2. This cell forms the initial syncytial cell (ISC). The dissolution of cell wall of ISC forms syncyntium multinucleate feeding structure. The process begins from plasmodesmata afterwhich the protoplasts of the adjoining cells fuse. The cells next to ISC go into syncytium repeatedly forming layers of the cells that become part of syncytium. DNA synthesis occurs making the metabolism rate to increase to give rich food for nematode (Bauters & Mohammad 2013, p. 380).
Nematode will remain in this stage for weeks after which it moults to adult stage. Females grow until the spherical bodies burst via the root surface while the males revert the vermiform bodies and leave the roots to follow the sex pheromone gradients of getting the females. The female dies after fertilization and her body wall tans and encloses the next egg generation (Bauters & Mohammad 2013, p. 381).
Figure 1.1: Swollen females for potato cyst nematode
(Photographs by CSL)
Damages Caused
Nematodes damage roots and lead to decreased yields even after the infestations lead to no serious symptoms within the haulm. For severe infestations, roots become more damaged and at times they may die. The severely infested plants have stunted growth, chlorotic and occur in patches. The rhizoctonia and other several fungal diseases that are associated with feeding of nematode also contribute to decreased yields (Aires & Rosa, 2009).
Figure 1.2: Syncytium induced by potato cyst nematode
Source: (Agriculture & Agri-Food Canada, Research Branch, 2001).
The potato cyst nematodes damage the roots resulting to decreased yields. The plant becomes prone to infestations that result in haulm. The severe root infestations damage the roots and may lead to eventual death of the plant. The infested plants have stunted growth, chlorotic and occur in patches. The rhizoctonia and several other fungal diseases contribute significantly to the loss of yields (Doyle & Kris 2002, p. 549).
ReniformNematode(RotylenchulusReniformis)
This is a sedentary endoparasite found in large number in perennial plant species. The pathogen cause widespread damages in crop, especially in tropical and sub tropical regions. This affects more than 350 plant species.
Lifecycle
The R. Reniformis has unique lifecycle. The 8-10-days old eggs hatch into J2s that undergo three successful moults without eating, and develop into adults (vermiform) males or females in 7-9 days. The adult adults are normally small than the J2s. The remained cuticles from the previous stages remain and they reduce water loss thereby improving anhydrobiotic survival rates in dry soil. Young females remain in infective stage where they penetrate through the plant roots inserting one third of their anterior bodies to form feeding sites on pericycle and endodermal cells. The feeding tube produced allows passage of all the materials ingested. After 2-3 days feeding, posterior section of the female body starts swelling during which it assumes a kidney-shape after one week. Uterine glands produce gelatinous matrix where 40-100 eggs are laid in 7-9 days. Males are normally numerous and they do not eat. Most become trapped in gravid females where they can be in entrapped in gelatinous matrix (Romero & Sandra 2005, p. 89).
Figure 1.3: Life cycle for Reniform Nematode
Source: (Field observations by UCNFA, 2011)
Structure of the research
i. To compare effector gene complement forRotylenchulusreniformisornamentals. and potato cyst nematode
This will involve taking the bioinformatics that will help compare putative effector genes and potentially identify novel effector genes from the R. reinformis. This will be carried out by blast searching of the predicted genes sequences for R. reinformis using the effector sequences that are known from the G. pallida as well as other nematodes.
ii. To characterise the nematode effector genes
Thereafter, a de novo analysis will be done for the predicted R. reniformis proteins to identify the novel genes with characteristics of effectors. This will involve use of a signal peptide for secretion without the transmembrane domain. The expression will be increased in parasitic stage without homology to the genes of identified non-effector function.
iii. Cloning of effector genes
A selection of the identified genes will be clones by collecting the nematodes and extracting RNA. This will enhance the synthesis of the cDNA through reverse transcriptase enzyme. The cDNA will act as template for PCR reactions with the designed primers to amplify complete coding of the region containing the four genes. The sequences for the amplified genes will be cloned into plasmid vectors and then sequenced to confirm that the predictions of thein silicoare correct.
iv. To assign an effector function to the gene
Assigning an effector function to the gene enhances determination the expression in the nematode. The effector proteins are secreted from oesophageal glands through nematode stylet into the host. This will be achieved through in situ hybridisation experiments to allow the determination of the expression for each of the four genes. For the effectors common to R.reniformis and G.pallida, the expression patterns will be compared for both species.
Justification of the resources requested
The researcher will undertake this study from the university. This is to enhance the acquisition of materials and resources from the university library, as well as allow quality research due to close guidance of the lecturers. The resources that will be required include the culture medium, reagents, cell lines, vectors and restriction enzymes. All the required equipment will be accessed from the university library. Any more materials required will be purchased through the department of biology.
Impacts of the proposed research
The major beneficiaries of the proposed research are farmers and agricultural research institutions. For instance, the focus on potato cyst nematode and Rotylrnchulusreniformisthat is important for many crops in countries that are warm. Any significant development regarding chroming and characterization of the parasitism nematodes will be communicated to scientific community to enhance publishing for easy access. The general public will also be enlightened through magazine and media press.
Ethical consideration
The research will involve taking bioinformatics approaches in comparing putative effector genes for Rotylrnchulusreniformis and potato cyst nematode. The process will involve various analysis using the effector sequences and de novo analysis to identify the novel genes with effectors` characteristics, as well ascloning of the novel effectors. The bioinformatics analysis will be carried out for transcriptome sequences, RNA/DNA extraction processes, culture of the parasitic nematode, in situ hybridisation, light microscopy and gene expression analysis using qCPR. Therefore, the research will not require any ethical permission.
References List
Aires, A., & Rosa, C. (2009). Suppressing Potato Cyst Nematode, Globodera Rostochiensis, with Extracts of Brassicacea Plants. American Journal of Potato Research 86(4), pp. 327-33.
Bauters, T., & Mohammad, M. (2013). Identification of Candidate Effector Genes in the Transcriptome of the Rice Root Knot Nematode. Molecular Plant Pathology 14(4), pp. 379-90.
Blaxter et al. (1998). Phylogeny of Wolbachia in Filarial Nematodes.” . Proceedings of the Royal Society B: Biological Sciences 265 (3), pp. 2403-413.
Doyle, E., & Kris, N. (2002). Cloning and Characterization of an Esophageal-Gland-Specific Pectate Lyase from the Root-Knot Nematode. Molecular Plant-Microbe Interactions 15(6), pp. 549-56.
Holterman, M. (2006). Phylum-Wide Analysis of SSU RDNA Reveals Deep Phylogenetic Relationships among Nematodes and Accelerated Evolution toward Crown Clades. Molecular Biology and Evolution 23(9), pp. 1792-800.
Romero, M. D., & Sandra, R. (2005). Soursop, a New Host of Rotylenchulus Reniformis. Fitopatologia Brasileira 30(4), pp. 89.
Senbergs, J., & Mark, J. (1998) CSL 70, 1916-1986: Commonwealth Serum Laboratories Commission the Seventieth Anniversary Exhibition: A Collection of Paintings, Drawings and Photographs by Jan Senbegs and Mark Johnson. Parkwood, Victoria: Commonwealth Serum Laboratories Commission.
Walker, B. D. (2001). Comparison of Cultivated and Native Soils in a Morainal Landscape in East Central Alberta Benchmark Site Comparison Report 05-AB vs. 55-AB ). Provost, Alberta : Ottawa: Agriculture and Agri-Food Canada, Research Branch.