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Molecular studies on the host parasite relationship of plants infected with the root-knot nematode Meloidogyne javanica

Ah-Fong, Marie Véronique Audrey (2000) Molecular studies on the host parasite relationship of plants infected with the root-knot nematode Meloidogyne javanica. PhD thesis, Murdoch University.

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Root-knot nematodes are serious economic pathogens of horticultural and crop plants in sub-tropical and tropical regions of the world including Australia. The nematodes invade the roots and are able to induce the redifferentiation of root cells into multinucleate feeding cells called 'giant cells'. These feeding cells form by repeated mitosis without cytokinesis and develop wall ingrowths typical of transfer cells.

It is clear that the pronounced morphological and physiological changes associated with the formation of these specialised feeding cells are caused by altered gene expression in host cells. For this reason, the study of molecular responses induced in host cells by the pathogen will not only provide new information to the host-parasite relationship but could provide better options for control strategies.

Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a tool to study changes in gene expression during the compatible interaction between tomatoes {Lycopersicon esculentum) and the root-knot nematode M javanica 25 days post-infection. A series of experiments were undertaken to develop this method, to improve sensitivity and reliability. Both short and long primers were used in the DD polymerase chain reaction (PCR). It was found that long primers in combination with a two-step PCR and the use of 33P as the radio-labelled nucleotide greatly improved the sensitivity and reproducibility of the technique. In addition, sequence-specific primers, which were originally designed for analysis of specific genes could be used as 5' arbitrary primers, further expanding the number of primers that could potentially be used in DD. Using a total of 16 primer combinations, 25 differentially expressed genes were identified, of which 12 were selected for further analysis.

Although DD has several advantages over other methods for isolating differentially expressed genes, it is also prone to artefacts. An efficient methodology was developed that enabled false positives to be discarded after the cloning step via a reverse Northern blot analysis. This involves probing the clones with labelled cDNAs from healthy and giant cell enriched tissues to differentiate between true and false positives. Clones harbouring truly differentially displayed bands were sequenced and semi-quantitative RT-PCR was used to reconfirm their differential expression and to allow isolation of low abundance differential cDNAs. Use of the screening strategy described here led to the isolation of six cDNAs; three down-regulated (HA/HT1 and HAS) and three upregulated (IC1, IC9 and IA2) genes whose expression was as predicted by the display gels, whilst minimising the amount of RNA used.

In order to determine the transcriptional activity of the genes more precisely, a relative real-time quantitative RT-PCR assay was developed that provides a quick, sensitive and reliable method of measuring and analysing expression patterns of genes. The starting materials from both healthy and giant cell enriched tissues were normalised to their relative amounts of actin in a TaqMan PCR assay. The expression pattern of the differentially expressed genes was then determined at various time points after infection (3, 15, 25 and 30 days) using SYBR Green I dye as the fluorescent dye, in TaqMan PCR. In healthy tissues, the expression pattern of the genes did not show any marked developmental regulation over time. However, significant changes in their transcriptional activity were observed in the giant cell enriched tissues particularly between 3 and 15 days after infection. Overall, the transcriptional regulation of the genes was up to 10-fold except for IC9 and HA/HT1, which showed a 69-fold increase and 28-fold decrease respectively, by 15 days post-infection. The real-time quantitative RT-PCR assay can be used to provide information on gene expression over the lifetime of the nematode plant association, and promises to be very widely applicable to study other systems. These results also demonstrate the sensitivity of DD to isolate genes that show subtle changes in gene expression.

Of the six differentially expressed genes studied in detail, five were found to be of plant origin and one (IC1) was found to be of nematode origin. Database searches of these differentially expressed genes revealed that at the amino acid level, the deduced IA2 gene product shares 72% identity to cucumber basic protein (a plantacyanin). HA3 and IC1 showed weak similarity to Arabidopsis thaliana transcription factor MYB59 and C. elegans cosmid C respectively. IC1 could be a nematode mRNA contamination or it could be part of the stylet component that the nematode injects inside the plant cells. If this results stands and it is not a contaminant of tissue preparation, the result is novel and could be highly significant. Transcripts HT1 and HA both showed very strong homology to the previously identified Lycopersicon esculentum RB7 gene and interestingly in this study, the level of its mRNA was found to be down-regulated during infection relative to control tissues. The full-length sequence of IC9 was determined by 5' RACE and its cDNA showed 58% identity with the gibberellin 2- oxidase protein from Piswn sativum, indicating the involvement of gibberellic acid in giant cell or gall formation. Because most of the genes identified represent the 3' noncoding regions of the mRNA or they are members of gene families for which the function is unknown, their significance of regulation is difficult to interpret without any further characterisation. Nevertheless, the results obtained are new, and add to the pattern of genes with altered expression in feeding cells.

The results presented in this thesis highlight the power of DD as a reliable, rapid and efficient method to systematically screen and identify differentially expressed genes in this system. The development of the SYBR Green PCR assay provides a novel approach to quantify native gene transcripts in giant cells compared to the use of a reporter gene assay. The genes isolated provide additional information on aspects of and function of nematode feeding cells. With the use of more primer combinations, many more genes transcriptionally regulated by the nematode can be revealed. This will lead to a better understanding of plant-nematode interactions and plant development, and may reveal ways to engineer sound and durable forms of synthetic resistance to the pathogen.

Item Type: Thesis (PhD)
Murdoch Affiliation(s): School of Biological Sciences and Biotechnology
Notes: Note to the author: If you would like to make your thesis openly available on Murdoch University Library's Research Repository, please contact: Thank you.
Supervisor(s): Jones, Michael and Potter, Robert
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