3. Inter´ es compuesto
4.3. Descuento comercial
4.3.2. Descuento comercial sucesivo
development of selective antibiotics
D. Phillips{ XE "Phillips, D." }1, D. Cahill2 and P.L. Beech11
Centre for Cellular and Molecular Biology, Deakin University, Burwood Vic 3125
2
Deakin University, Waurn Ponds Vic 3216
Phytophthora is probably best known for causing the Irish potato
famine of the 1840s, but this plant pathogen is not just an issue
of history. Recent estimates show P.sojae to cause $1–2 billion in
soy bean and $400 million in tomato crop losses p.a., and that P.infestans costs ~$290 million p.a. in management and losses of
potato crops in the USA (e.g. 1). In Australia, P. cinnamomi is one
of the greatest risks to our terrestrial ecosystems: it destroys
numerous non‐arid habitats, having a wide host range of up to
2500 plant species (2), and is thus considered a key threat under
the Commonwealth Environmental Protection and Biodiversity
Conservation Act 1999. Development of a control for Phytophthora spp. is critical to future sustainable agriculture and
will be invaluable in maintaining numerous ecosystems in
Australia and abroad. This project aims to directly target
Phytophthora in the same manner used to develop the anti‐
influenza drug, Relenza⎢(3) by identifying and solving the
structure of a protein(s) unique to and critical for the survival of
Phytophthora, we aim to provide the information required for
future development of customised antibiotics.
Only a handful of biological components are so critical to life that
they are conserved throughout all organisms. For example,
ribosomes are required for protein synthesis and as such are
found in every living cell. So too, several signaling cascade
enzymes appear to have been conserved across life;
phospholipase C (PLC) is one such enzyme conserved from
bacteria to humans and, just as the loss of ribosomes would be
fatal, the loss of PLC would be catastrophic to the cell. How is it
then, that plant pathogens of the genus Phytophthora do not
have any recognizable PLC (4)? Phospholipase C is a transient
membrane protein that, upon GTP activation, hydrolyses the
phospholipid phosphoinositide bisphosphate (PIP2) into the
secondary messengers (1,4,5) inositol triphosphate (IP3) and
diacylglycerol (DAG), which inter alia activate protein kinase
pathways, phospholipase D pathways and mediate rapid calcium
release from the endoplasmic reticulum (5).
We propose that PLC has been replaced by an alternative
protein we call AltPLC. Existence of such an alternate protein
would not only represent significant insight into the evolution of Phytophthora, but may indeed represent an ideal target for anti‐
Phytophthora antibiotics.
We have approached the problem of identifying the AltPLC from
three directions, utilising structural bioinformatics, differential
proteomics, and biochemical analysis.
METHODS
Structural bioinformatics: We have identified all proteins within
the P. sojae genome which bind to PIP2 using Hidden Markov
Models to search for patterns which convey the structure of
Plekstrin homology domains –a structure known to specifically
bind to PIP2. This data set was then scrutinised by a number of
domain‐architecture mapping and structural prediction
algorithms.
Differential proteomics: we have developed a method of
isolating transient membrane proteins. This involves cracking the
cells under high calcium and low temperature conditions, to bind
lipid‐regulating proteins to the membrane fragment. After
washing the membrane, elution is achieved by removing Ca2+
and allowing dissociation. Analysis of these fractions was
performed by MS/MS and in vitro hydrolysis reactions.
Biochemical analysis: IP3 was isolated using the method of Lorke et al. (2004)(6) and analyzed by MDD‐HPLC (7).
RESULTS
Using MDD‐HPLC we have shown that P. cinnamomi does
produce IP3 endogenously and DAG in vitro by hydrolysis
reactions with differentially isolated transient membrane protein
fractions. This evidence supports our hypothesis of an
alternative PLC in the Phytophthora genus. Using our
combinatorial bioinformatics approach we have uncovered a
single protein with all necessary structural components to
perform PIP2 hydrolysis. Furthermore, this protein is conserved
among P. sojae P. ramourum and P. infestans and, as
hypothesised, is unique to the Phytophthora genus. We have
cloned and continue to isolate, recombinant AltPLC and its
activator RAS protein for functional and structural analysis.
although final correlation between PIP2 hydrolysis and our
putative AltPLC protein has yet to be achieved. Beyond the
obvious development of novel control methods, identification of
a phospholipase C protein of independent evolutionary origin is
a unique and significant discovery that may ultimately aid in
elucidating / refining the evolutionary origins of Phytophthora,
and give us an insight into the process of independent
convergence events in general terms.
REFERENCES
1. Tyler,B.M., Henkart,M. (2005) Genome information from plant destroyers could save trees, beans and chocolate. National science
foundation press.
http://www.nsf.gov/news/news_summ.jsp?cntn_id=107973&org= NSF&from=news
2. Hardham, A. (2005) Phytophthora cinnamomi. Molecular Plant
Pathology 6: 589–604
3. Coleman,P,M., Hoyne,P,A., Lawrence,M,C. (1983) Sequence and
structure alignment of paramyxovirus hemagglutinin‐
neuraminidase with influenza virus neuraminidase. Journal of Virology 67: 2972–2980
4. Tyler BM. et al. (2006) Phytophthora genome sequences uncover evolutionary origins and mechanisms of pathogenesis. Science 313:
1261–1266.
5. Durrell,J. Sodd,M,A. Friedel,R.O. (1968) Acetylcholine stimulation of
phosphodiesteratic cleavage of the guinea pig brain
phosphoinositides. Journal of Life Science 7: 363–368
6. Lorke DE. Gustke H, Mayr GW (2004) An Optimized Fixation and Extraction Technique for High Resolution of Inositol Phosphate Signals in Rodent Brain Neurochemical Research, Vol. 29, No. 10, pp. 1887–1896
7. Mayr GW (1988) A novel metal‐dye detection system permits picomolar‐range h.p.l.c. analysis of inositol polyphosphates from non‐radioactively labelled cell or tissue specimens Biochem. J. (1988) 254, 585–591
Session
4A—Plant
pathogen
interactions
Systemic acquired resistance—a new addition to the IPM clubroot toolbox?
A. AgarwalA, E.C. Donald{ XE "Donald, E.C." }A, R. FaggianB, D.M. CahillC, D. LovelockC and I.J. PorterAA
Department of Primary Industries Victoria, Private Bag 15, Ferntree Gully Delivery Centre, 3156, Victoria
B
Department of Primary Industries Victoria, 32 Lincoln Square Nth, Carlton, 3053, Victoria
C
Deakin University, School of Life and Environmental Sciences, Waurn Ponds Campus, Geelong, 3217, Victoria
INTRODUCTION
Clubroot caused by Plasmodiophora brassicae affects the
Brassicaceae family of plants causing root galling, stunting and
wilting of many important vegetable crops. There has been no
‘silver bullet’ solution to clubroot but a number of ‘tools’ are
available to manage the disease. Integrated use of these ‘tools’,
including detection of P. brassicae and prediction of yield loss
due to clubroot, identification and elimination of hygiene risks
together with in‐field cultural methods, use of resistant varieties,
manipulation of soil pH, calcium and boron amendment and
strategic use of pesticides has been extremely effective in
vegetable production systems (1).
Microarray analysis conducted at the early time points during
the infection process of P. brassicae in Arabidopsis (4, 7 and 10
days after inoculation) identified a number of genes and
pathways that may regulate disease expression in Arabidopsis
(2). Manipulation of the salicylic acid (SA) signalling pathway may
induce systemic acquired resistance (SAR), a state of heightened
defensive capacity in plant species. This paper describes
preliminary experiments to study the effect of SA as an inducer
of SAR in Arabidopsis and broccoli, and assess the potential for
SAR to be incorporated into the IPM ‘toolbox’ for clubroot
management.
MATERIALS AND METHODS
A proof of concept study was conducted using Arabidopsis.
Roots were treated with 0.5 mM SA for 1 minute and then
inoculated with P. brassicae resting spores 4 hours after
treatment. Plants were assessed for disease expression 50 days
after inoculation.
A broader range of SA dip rates (1–10 mM) and contact times
were evaluated in order to induce SAR in broccoli. Plants were
inoculated with a spore suspension of P. brassicae 24 hours after
treatment and assessed for disease expression 6 weeks after
inoculation. A real‐time reverse transcriptase quantitative PCR
(RT‐qPCR) assay was developed to determine the expression of
the chitinase gene in broccoli roots and leaves. Biochemical
methods are also being developed to confirm SAR induction.
RESULTS AND DISCUSSION
Clubroot disease was strongly suppressed in salicylic acid treated Arabidopsis plants (Fig 1). Fifty days post‐inoculation SA treated
plants had a much lower disease index and infection rate (DI=20,
IR=50%) compared to untreated plants (DI=81.5, IR=100%).
A 15 min root dip in 1 mM SA 24 hours before inoculation was
the most effective method of SAR induction in broccoli. This
treatment consistently increased expression of the chitinase
gene by between 2.3 and 5.5 fold in roots and leaves confirming
a systemic response. At concentrations in excess of 1 mM SA,
changes in the expression of the chitinase gene were less
consistent. Frequently these higher concentrations of SA caused
a decrease in the expression of the chitinase gene. At the higher
rates SA might not be translocated or it may alter the physiology
of the plant. A similar result (ie. increased control only at the
lowest rate 1 mM) was obtained from disease expression studies
using broccoli (Fig 2). SA was phytotoxic to plants at 10 mM.
Figure 1. Arabidopsis plants 50 days after inoculation with P. brassicae.
Plants on the right have been pretreated with salicylic acid to induce SAR.
Figure 2. Symptoms of clubroot on roots of broccoli plants 6 weeks after
inoculation which occurred 24 hrs after treatment with SA (clockwise from top left 0, 1, 2.5 and 10 mM SA). Each image shows the range of symptoms in each treatment group.
This is the first evidence that SAR induction may be a useful
addition to the IPM clubroot ‘toolbox’. Work is ongoing to
further optimise rates and timing of application of SA, to identify
and evaluate other inducers and to extend the work to other
pathogens.
ACKNOWLEDGEMENTS
This work has been funded by DPI Victoria and Horticulture
Australia Limited (HAL) using the vegetable levy and matched
funds from the Australian Government.
REFERENCES
1. Donald C, Porter I (2009) Integrated control of clubroot. Journal of
Plant Growth Regulation (In Press). doi: 10.1007/s00344‐009‐9094– 7
2. Agarwal A (2009) Interactions of Plasmodiophora brassicae with Arabidopsis thaliana. PhD thesis, Deakin University.