Phosphite has been used to effectively control the soil borne plant pathogen Phytophthora cinnamomi in many horticultural crops, forest trees and natural ecosystems. However, the molecular mechanisms behind phosphite action on this pathogen are poorly understood.
Several studies have shown that phosphite inhibits growth and zoospore production of P. cinnamomi and in addition induces significant physiological and metabolic changes in the mycelium. As an approach to understanding the mechanisms and relevance of these changes in the pathogen, the effect of phosphite on gene expression was investigated using microarray analysis. To construct the microarray, RNA was extracted from phosphite-treated (40 ug/ml) mycelium of P. cinnamomi isolate MP 80. The chosen phosphite concentration inhibited the mycelial growth by 70% but provided sufficient mycelium for RNA extractions after 4 days growth at 25C.
The mRNA was reverse transcribed into cDNA and cloned into lambda to construct a library consisting of 2 million pfu of which 80 % were recombinant phage. The inserts were sequenced for a random selection of clones from the library. The nucleotide sequences generated revealed a range of different P. cinnamomi genes being expressed and demonstrated that the cDNA library provided a good representation of the transcripts expressed in P. cinnamomi. The types of genes found to be expressed in the mycelium of P. cinnamomi included genes encoding GTP binding proteins involved in vesicle transport, structural proteins involved in maintaining cell membrane integrity,elicitors, phosphatases and ribosomal proteins.
Over nine thousand cDNA transcripts were randomly selected from the cDNA library and prepared by PCR amplification and purification for microarray construction. Custom made cDNA arrays containing 9216 cDNA transcripts were constructed and probed with RNA from untreated mycelium and mycelium grown in medium with 40 ug/ml phosphite.
Two genes, EF-1 alpha and cinnamomin gene, identified by qRT-PCR as being constitutively expressed were also positioned on the arrays as positive controls. In the process of identifying constitutively expressed genes, qRT PCR revealed that phosphite down-regulated a gene encoding ubiquitin-conjugating enzyme, a component of the ubiquitin/proteasome pathway involved in the removal of abnormal and short lived-regulatory proteins and rate limiting enzymes.
From the arrays a further seventy-two transcripts with altered patterns in gene expression (fold change > 2) were identified. The majority of the cDNA transcripts spotted on the array were down-regulated with changes in gene expression ranging from 2- to 3.5-fold. Thirty-two cDNA transcripts were up-regulated with changes in gene expression ranging from 2- to 16-fold. Characterisation by sequencing revealed that the most highly induced transcripts coded for ADP-ribosylation factors, an ABC cassette transporter and a glycosyl transferase. A transcript encoding a vitamin B6 biosynthesis protein was also identified as up-regulated by 2.9-fold. In contrast, the down-regulated transcripts coded for cellulose synthase I, annexin, glutamine synthetase, metallothionein and an alternative oxidase. The results are discussed in terms of possible roles and mechanism(s) of phosphite action within the mycelium of P.cinnamomi.
This work is the first comprehensive screen for phosphite regulated-gene expression in P. cinnamomi and represents a significant step towards an understanding of the mode of action of phosphite on this organism. This thesis provides valuable information on the molecular interaction between phosphite and P. cinnamomi, which in future studies may stimulate the discovery of novel methods and cellular targets for the control of plant pathogenic Oomycetes.
| Identifer | oai:union.ndltd.org:ADTP/178840 |
| Date | January 2007 |
| Creators | M.King@murdoch.edu.au, Michaela King |
| Publisher | Murdoch University |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | http://www.murdoch.edu.au/goto/CopyrightNotice, Copyright Michaela King |
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