Molecular studies on gene expression in host plants infected with the root-knot nematode Meloidogyne javanica

Root-knot nematodes are economically important phytopathogenic endoparasites that invade more than 2,000 species of horticultural and crop plants in sub-tropical and tropical regions of the world, including Australia. Infection by root-knot nematodes induces the redifferentiation of normal provascular cells of the host roots into multinucleate feeding cells called ‘giant cells’, which are surrounded by a gall. Giant cells form by repeated mitosis without cytokinesis, and developing wall ingrowths typical of transfer cells. Giant cells act as sources of nutrients for the development of the nematode parasite, and break down shortly after the nematode has completed its life cycle. The pronounced morphological and physiological changes associated with infection by the nematode, especially in giant cells, are the result of altered gene expression in host root cells. Since giant cells are the only root tissue from which the nematode can feed, these giant cells are the subject of much molecular investigation on plant-nematode interactions. The study of gene expression in giant cells will provide new information on the hostparasite relationship, and lead to novel strategies for engineering host plant resistance. mRNA differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used to study changes in gene expression in giant cells during the compatible interaction between tomato (Lycopersicon esculentum) and the root-knot nematode Meloidogeny javanica at 25 days post-inoculation. Methods of direct extraction of cytoplasmic contents from individual giant cells were developed using a modified pressure probe system. The giant cell origin of the extracted cytoplasm was confirmed by the presence of multiple nuclei in extracts after staining with different fluorescent dyes. mRNA was isolated from the extracted giant cell cytoplasm using magnetic Dynabeads, and its use to study gene expression in giant cells was evaluated by RT-PCR analysis. A series of experiments were undertaken to improve the sensitivity and reliability of DDRTPCR in the gene expression studies. With a total of 44 primer combinations, 81 differentially displayed bands were isolated from differential display (DD) gels. Although DDRT-PCR offers several advantages over other methods for the isolation of differentially expressed genes, it can also lead to artifacts. An efficient method was developed to identify true up- or down-regulated genes from a relatively large number of DD bands being analysed. This involved single pass direct sequencing of the re-amplified DD bands, with the same anchor or arbitrary primers used in differential display reactions to generate these DD bands. Of the 81 DD bands, 27 produced readable sequences, of which 17 were selected for further analysis. Sequence specific primers for these 17 DD bands were designed and used to carry out real-time quantitative RT-PCR to re-confirm the differential expression of these genes and relatively quantify it in giant cells. The differential expression of the 16 genes, 15 up-regulated and 1 down-regulated in giant cells, were successfully re-generated by the quantitative RT-PCR assay. The expression of one gene, ZW30050025, was only detected in giant cell cytoplasmic contents but not in non-infected control, and so could be giant cell specific. Further study of this gene needs to be undertaken, since it could be used in engineering plant resistance. Of the 17 differentially expressed genes, transcriptional regulation of ZW2703003 showed about 56-fold increase in giant cells compared to healthy root tissue at 25 days postinoculation, and a 10-fold decrease for ZW1307002. However, in analysis of the time course of expression of these two genes, a dilution effect on the regulation of gene expression was observed when giant cell enriched tissue was used as starting material. These results indicate that the use of giant cell cytoplasmic contents will provide more accurate information on differential gene expression in giant cells, and so help understanding of the plant-nematode interaction. The functions of the differentially expressed genes in maintenance and development of giant cells were predicted by database similarity searches. At the amino acid level, the deduced products of several genes, ZW0805001, ZW0903001, ZW0903002 and ZW30050020, shared strong identities with different types of ribosomal proteins. The upregulation of these ribosomal protein genes, which in turn reflects the high translation activities, agrees with the predicted high metabolic activity in giant cells. Similarly, other genes encoding proteins with homology to those involved in high metabolic activity were also found to be up-regulated in giant cells. These genes included a Histone H3 gene (ZWO103005), a S-adenosylmethionine decarboxylase (SAMDC) gene (ZW0103003), a cysteine synthase gene (ZW0103001), and a cytochrome c reductase hinge protein subunit gene (ZW3107005). By using 5’ RACE, more sequence information of the coding region of genes ZW2703003, ZW1008005 and ZW1307002 was obtained. The deduced amino acid sequence of ZW 1008005 showed 40% identity over 146 amino acid residues with the unique C-terminal region of TDY (Thr-Asp-Tyr) type mitogen-activated protein kinase (MAPK), indicating a complex single transduction pathway involved in the function of giant cells. The down-regulated gene identified in this study, ZW1307002, shared very weak identity with metallocarboxypeptidase inhibitor (MCPI). The real role of ZW1307002, as well as the high up-regulated gene ZW2703003, remains to be determined by further investigation. Other genes identified in this study to be up-regulated included one gene probably involved in the pathogen responses, a cytosolic glyceraldehyde-3- phosophate dehydrogenase (GAPDH) gene (ZW0103004). Although further analysis is necessary to understand the significance of the altered regulation of these genes in relation to giant cell function, the results obtained in this study are new, and add to the known pattern of genes with altered expression in nematode feeding cells. The results presented in this thesis indicate that extracted cytoplasmic contents can be used as an appropriate starting material in DDRT-PCR analysis to identify differentially expressed genes in giant cells. The development of direct sequencing combined with realtime quantitative RT-PCR assay provides an efficient approach to verify changes of the transcripts level in giant cells, particularly when a large number of DD bands are being analysed. With more genes isolated and identified, a better understanding of the molecular events in giant cells in the plant-nematode interactions will be achieved, which should contribute to the development of new strategies to provide plant resistance against these pathogens