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Abstract

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Therehas been tremendous advancement in analysis and identification of thefunctional nematode effector genes. At the same time, research hasbeen emphasized on cloning and characterization of the nematodeparasitism genes of effector proteins. These are normally secretedthrough the stylet of the nematode that promotes nematode parasitismin plants. To date, cloning has been achieved for at least 100parasitism genes while host functions and targets for the secretedeffector genes have been elucidated. Therefore, the current researchwill seek to identify the gene sequences for the effector dependingon profile expression, the prediction of signal transmembrane/peptidedomain and comparison with the other nematode species. The identifiedgenes will be cloned using qPCR and experiments will be used toidentify the spatial expression patterns using insitu hybridisationprocess.

Theplant-parasitic nematodes form the biotrophic parasites that feedfrom cytoplasm of the unmodified living cells of a plant. Thenematodes modify the cells into discrete feeding cells to enable themobtain nutrients that are necessary for growth and development. Thecurrent project will identify the effector genes sequences dependingon the expression profiles and the identified genes will be clonedusing qPCR while the expression will be identified using insitu hybridisation.The project will help in determining the expression for putativeeffector genes. This will help in generating a transcriptome assemblyfor the nematode to conform to the silicogene expressions.

Figure1.0: Schematicrepresentation of phylum nematode with examples of nematode speciesin brackets and parasite species shown for each branch

Source(Holterman et al. (2006) and Blaxter et al. (1998))

ProblemStatement

Theevolution of plant parasitic nematodes can be traced back to thebiotrophic interactions with the host. The secretion from nematodegland penetrates the root cells through the stylet resulting intoprofound changes within the gene expression of the plant and the cellstructure. The transcriptomes have recently been sequenced for threeplant parasitic nematodes.

Aimsand objectives of the research

  1. To assign an effector function to the gene

  2. Cloning of effector genes

  3. To characterise the nematode effector genes

  4. To compare effector gene complement for Rotylenchulusreniformis and potato cyst nematode

Backgroundto the research

Overthe last one decade, there has been extensive research on parasiticnematode effector genes. Such surveys have triggered discussions onpathogens and their economic effects. The plant parasitic nematodesportray a wide variety of the parasite-host interactions. All of themcontain a stylet that penetrates the cells to allow feeding. Some ofthe nematodes are migratory ectoparasites while others are migratoryendoparasites. Nevertheless, cyst and root-knot nematodes are themost economically important and are biotrophic in nature. There areover 4100 parasitic nematodes but this research will focus onRotylrnchulusreniformisandpotato cyst nematode (Aires &amp Rosa 2009, p. 327).

PotatoCyst Nematodes(Heterodera and Globodera Spp.)

Cystnematodes form the obligate biotrophs that have great economicimportance worldwide. The soybean cyst nematodes (SCNs) are the mostdamaging 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 &ampRosa 2009, p. 328). The major problem is that the nematodes cansurvive in soil for prolonged periods and this makeseradicationdifficult (Bauters &amp Mohammad 2013, p. 379).

Lifecycle

Cystnematodes moult into an egg in J2 stage. This stage is dormant and ishost-specific. Hatching occurs based on the host-derived chemicalcues that are present in the root diffusates (Perry, 2002). J2locates its host by invading and migrating intracellularly anddestructively through the root into the inner cortex where thebehaviour changes. J2 inserts the stylet into the root cells andwaits for the response of the cell. The stylet retracts when it iscovered with cellulose layer or after bursting of the protoplasm.This occurs repeatedly until it reaches a cell that is not adverselyaffected by J2. This cell forms the initial syncytial cell (ISC). Thedissolution of cell wall of ISC forms syncyntium multinucleatefeeding structure. The process begins from plasmodesmata afterwhichthe protoplasts of the adjoining cells fuse. The cells next to ISC gointo syncytium repeatedly forming layers of the cells that becomepart of syncytium. DNA synthesis occurs making the metabolism rateto increase to give rich food for nematode (Bauters &amp Mohammad2013, p. 380).

Nematodewill remain in this stage for weeks after which it moults to adultstage. Females grow until the spherical bodies burst via the rootsurface while the males revert the vermiform bodies and leave theroots to follow the sex pheromone gradients of getting the females.The female dies after fertilization and her body wall tans andencloses the next egg generation (Bauters &amp Mohammad 2013, p.381).

Figure1.1:Swollen females for potato cyst nematode

(Photographsby CSL)

DamagesCaused

Nematodesdamage roots and lead to decreased yields even after the infestationslead to no serious symptoms within the haulm. For severeinfestations, roots become more damaged and at times they may die.The severely infested plants have stunted growth, chlorotic and occurin patches. The rhizoctonia and other several fungal diseases thatare associated with feeding of nematode also contribute to decreasedyields[ CITATION Air09 l 1033 ].

Figure1.2: Syncytiuminduced by potato cyst nematode

Source:(Agriculture&amp Agri-Food Canada, Research Branch, 2001).

Thepotato cyst nematodes damage the roots resulting to decreased yields.The plant becomes prone to infestations that result in haulm. Thesevere root infestations damage the roots and may lead to eventualdeath of the plant. The infested plants have stunted growth,chlorotic and occur in patches. The rhizoctonia and several otherfungal diseases contribute significantly to the loss of yields (Doyle&amp Kris 2002, p. 549).

ReniformNematode(RotylenchulusReniformis)

Thisis a sedentary endoparasite found in large number in perennial plantspecies. The pathogen cause widespread damages in crop, especially intropical and sub tropical regions. This affects more than 350 plantspecies.

Lifecycle

TheR. Reniformis has unique lifecycle. The 8-10-days old eggs hatch intoJ2s that undergo three successful moults without eating, and developinto adults (vermiform) males or females in 7-9 days. The adultadults are normally small than the J2s. The remained cuticles fromthe previous stages remain and they reduce water loss therebyimproving anhydrobiotic survival rates in dry soil. Young femalesremain in infective stage where they penetrate through the plantroots inserting one third of their anterior bodies to form feedingsites on pericycle and endodermal cells. The feeding tube producedallows passage of all the materials ingested. After 2-3 days feeding,posterior section of the female body starts swelling during which itassumes a kidney-shape after one week. Uterine glands producegelatinous matrix where 40-100 eggs are laid in 7-9 days. Males arenormally numerous and they do not eat. Most become trapped in gravidfemales where they can be in entrapped in gelatinous matrix (Romero &amp Sandra 2005, p. 89).

Figure1.3: Lifecycle for Reniform Nematode

Source:(Field observations by UCNFA, 2011)

Structureof the research

  1. To compare effector gene complement forRotylenchulusreniformisornamentals. and potato cyst nematode

Thiswill involve taking the bioinformatics that will help compareputative effector genes and potentially identify novel effector genesfrom the R. reinformis. This will be carried out by blast searchingof the predicted genes sequences for R. reinformis using theeffector sequences that are known from the G. pallida as well asother nematodes.

  1. To characterise the nematode effector genes

Thereafter,a denovo analysiswill be done for the predicted R. reniformis proteins to identifythe novel genes with characteristics of effectors. This will involveuse of a signal peptide for secretion without the transmembranedomain. The expression will be increased in parasitic stage withouthomology to the genes of identified non-effector function.

  1. Cloning of effector genes

Aselection of the identified genes will be clones by collecting thenematodes and extracting RNA. This will enhance the synthesis of thecDNA through reverse transcriptase enzyme. The cDNA will act astemplate for PCR reactions with the designed primers to amplifycomplete coding of the region containing the four genes. Thesequences for the amplified genes will be cloned into plasmid vectorsand then sequenced to confirm that the predictions of theinsilicoarecorrect.

  1. To assign an effector function to the gene

Assigningan effector function to the gene enhances determination theexpression in the nematode. The effector proteins are secreted fromoesophageal glands through nematode stylet into the host. This willbe achieved through insitu hybridisation experimentsto allow the determination of the expression for each of the fourgenes. For the effectors common to R.reniformis and G.pallida, theexpression patterns will be compared for both species.

Justificationof the resources requested

Theresearcher will undertake this study from the university. This is toenhance the acquisition of materials and resources from theuniversity library, as well as allow quality research due to closeguidance of the lecturers. The resources that will be requiredinclude the culture medium, reagents, cell lines, vectors andrestriction enzymes. All the required equipment will be accessed fromthe university library. Any more materials required will be purchasedthrough the department of biology.

Impactsof the proposed research

Themajor beneficiaries of the proposed research are farmers andagricultural research institutions. For instance, the focus on potatocyst nematode and Rotylrnchulusreniformisthatis important for many crops in countries that are warm. Anysignificant development regarding chroming and characterization ofthe parasitism nematodes will be communicated to scientific communityto enhance publishing for easy access. The general public will alsobe enlightened through magazine and media press.

Ethicalconsideration

Theresearch will involve taking bioinformatics approaches in comparingputative effector genes for Rotylrnchulusreniformisand potato cyst nematode. The process will involve various analysisusing the effector sequences and denovo analysisto identify the novel genes with effectors’ characteristics, aswell ascloning of the novel effectors. The bioinformatics analysiswill be carried out for transcriptome sequences, RNA/DNA extractionprocesses, culture of the parasitic nematode, insitu hybridisation,light microscopy and gene expression analysis using qCPR. Therefore,the research will not require any ethical permission.

ReferencesList

Aires, A., &amp 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., &amp 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.

&nbspBlaxter et al. (1998). Phylogeny of Wolbachia in Filarial Nematodes.&quot . Proceedings of the Royal Society B: Biological Sciences 265 (3), pp. 2403-413.

Doyle, E., &amp 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., &amp Sandra, R. (2005). Soursop, a New Host of Rotylenchulus Reniformis. Fitopatologia Brasileira 30(4), pp. 89.

Senbergs, J., &amp 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.