More than 100 new plant signaling peptides identified

Unlike animals, plants can’t run away from their environment or other organisms. They contain hundreds of receptors to monitor their environment. The receptor ligands however, are largely unknown. Especially so called leucine-rich repeat receptor-like kinases (LRR-RLKs) are much more abundant in plants than in animals. Motivated through the involvement of LRR-RLKs in the symbiotic interaction between rhizobial soil bacteria and leguminous plants, such as pea, bean and soybean as well as the potential involvement in the infection of plant roots by parasitic nematodes we identified more than 100 new signaling peptides that are interaction partners of this predominant plant receptor family. Their sequences correlated with their functional properties and functionally similar signaling peptides clustered into groups. Furthermore, we identified conserved and invariant residues in the region of the active signaling peptide that might mediate receptor interactions or even determine receptor specificity. We also identified untypical members of the family carrying several active signaling peptides in their protein precursor instead of a single signaling peptide. Finally, newly identified signaling peptides were tested functionally and we demonstrated that their action on plant growth is sequence specific.

Oelkers K, Goffard N, Weiller GF, Gresshoff PM, Mathesius U, Frickey T.
Bioinformatic analysis of the CLE signaling peptide family.
BMC Plant Biol. 2008 Jan 3;8:1.
[Abstract at PubMed] [Fulltext at BioMed Central]

microRNAs; master regulators of gene activity and development.

It was once thought that all the important regulatory functions of higher organisms were performed by proteins, and that the DNA between protein-coding genes was nothing other than junk DNA. As it turns out there are many important genes in this so-called “junk”, where they code for RNA structures (see Figure A) that are processed into small 21 nucleotide molecules (Fig B) known as microRNAs (miRNAs). With our CSIRO colleagues, we have isolated a mutant in which the model plant Arabidopsis fails to produce one type of miRNA known as miR159. This has dramatic consequences for the plant’s development, where the miRNA mutant develops curled leaves, smaller fruits and smaller seeds. These traits are a consequence of the de-regulation of a family of genes that code for transcription factors known as GAMYBs, where each family member has a sequence to which miR159 can bind to and then destroy the gene’s transcript (mRNA). This regulation is shown with a reporter gene (gives a blue colour) that has been joined to a GAMYB gene; in the miRNA mutant there is strong activity (indicating loss of regulation), whereas in the normal wild-type plant no activity can be detected, indicating the gene is being naturally “silenced”. Correlating this gene activity with the mutant traits demonstrates that this natural gene silencing mechanism is critical for proper plant development. Understanding this recently discovered form of gene regulation may provide insights on how to manipulate leaf shape, fruit and seed size, all extremely important agronomic traits.

Allen, RS, Li J, Stahle MI, Dubroué A, Gubler F, Millar AA (2007) Genetic analysis demonstrates functional redundancy and the major target genes of the Arabidopsis miR159 family. Proceedings of the National Academy of Sciences, USA (in press).

The uptake and metabolism of a shampoo ingredient by malaria parasites.

We have previously shown (see earlier Research Breakthrough below called “Killing malaria parasites by starving them of vitamin B₅)that a common shampoo ingredient called pantothenol, kills the red blood cell stage of malaria parasites by cutting off their supply to the essential nutrient vitamin B5.  In this study we show that the intracellular parasite takes up pantothenol via distinct mechanisms across the red blood cell membrane and the parasite membrane.  More importantly, we show that the parasite then metabolises pantothenol by the same initial process it uses to metabolise vitamin B5, and that this process is under tight regulatory control by the parasite.  Furthermore, we discovered that this tight regulation can be inhibited by a compound called furosemide (which is used clinically to treat hypertension and certain types of edema).  Our study therefore, not only provides additional information on the mechanism by which pantothenol kills the parasite but also demonstrates that the process regulating vitamin B₅ metabolism can be disrupted by small molecule inhibitors and may therefore represent a new target by which to kill the parasite.

Transporter or Channel?

Transporters and channels are considered to operate in a different way. A channel is like an open door in the membrane, whereas transporters are likened to sliding doors with swipe card access. Stefan Bröer’s group together with German colleagues have studied a transporter which also behaves like a channel. The protein is involved in the release of the neurotransmitter precursor glutamine from brain astrocytes. Transport properties were studied by microelectrodes, which detect changes of the membrane potential and of the intracellular pH. The studies revealed a pore in the transporter that can be used by glutamine itself but also by ions in a similar way as they move through an ion channel.

Schneider, HP, Broer, S, Broer, A, and Deitmer, JW (2007) Heterologous expression of the glutamine transporter SNAT3 in Xenopus Oocytes is associated with four modes of uncoupled transport. J Biol Chem 282: 3788-98

Details can be found at: http://www.jbc.org/cgi/content/full/282/6/3788

How the malaria parasite gets an essential amino acid

Amino acids are the building blocks of proteins. As it grows within its host red blood cell the malaria parasites utilizes large quantities of amino acids for this purpose. The malaria parasite derives most of its amino acid requirements from chopping up the haemoglobin of its host blood cell. But haemoglobin lacks one important amino acid, isoleucine, and the parasite therefore has to source this from elsewhere. In this study we have shown that isoleucine is taken up into the infected blood cell from the external environment (the blood plasma), primarily via novel channel induced by the parasite in the host red blood cell membrane. Having entered the infected cell isoleucine is rapidly taken up into the parasite via a sophisticated ‘transporter’ mechanism that allows the parasite to take up isoleucine in exchange for another amino acid (leucine) which is the most abundant amino acid in haemoglobin and which the parasite therefore has in abundance. The transport pathways involved in isoleucine uptake play a crucial role in the parasite and are potential antimalarial drug targets.

Martin, R.E. and Kirk, K. (2007) Transport of the essential nutrient isoleucine in human erythrocytes infected with the malaria parasite Plasmodium falciparum. Blood, Prepublished online October 17, 2006; DOI 10.1182/blood-2005-11-026963 [Pubmed]

Improving drought tolerance

Stresses such as excess light and drought reduce plant yield and can be lethal. We are identifying the mechanisms by which plants perceive and respond to drought and excess light.

The approach utilizes cutting-edge genomic technologies, such as DNA chip arrays and high throughput screening of promoter-reporter gene fusions to identify novel genes and signalling pathways in the model plant, Arabidopsis. We have identified “gain-of-function” mutants that withstand drought.

Ultimately, an understanding of these mechanisms will be incorporated into traditional plant breeding programs towards improving crop yield.

Survival and optimal growth of mutants in the green alga Chlamydomonas under extreme light and oxidative stress

Even though photosynthesis depends on absorption of light energy to fix inorganic carbon for growth and productivity in plants and in virtually all other photosynthetic organisms, too much of a good thing becomes detrimental. Prolonged exposure to full sun light exceeds the capacity of the photosynthetic apparatus to dissipate excess absorbed light energy as harmless heat, and hence increases the generation of reactive oxygen species (ROS) likely to damage protein, lipids and DNA, ultimately causing cell death. We isolated spontaneous mutants (VHL^R) in the green model alga Chlamydomonas reinhardtii that are surprisingly capable of growing in continuous full sun light, and to tolerante elevated levels of a range of ROS. Detailed physiological and biochemical characterizations revealed that this VHL^R trait is a “gain of function” without any growth penalty, unlike the frequently observed decrease in yield as in various herbicide resistant or abiotic stress resistant mutants. The Chlamydomonas mutants maintain high levels of photosynthetic function even under extreme conditions. Chloroplast function is improved by altering the activity and effectiveness of several photosynthetic, photoprotective and other metabolic function in the chloroplast simultaneously. Those physiological responses are accompanied by a largely altered proteome profile, clearly indicating that VHL resistance is the result of pleiotropic effects of a mutation in a single nuclear gene. We hypothesize that VHL^R genes are regulatory “master switches” that coordinate expression of individual photoprotective and light stress response processes. Our current research is focused on positional, map-based cloning to identify one of the VHL^R genes, as the next crucial step to a mechanistic understanding of high light stress in plants.

Förster B., Mathesius U., Pogson B.J. (2006) Comparative proteomics of high light stress in the model alga Chlamydomonas reinhardtii. Proteomics 6: 4309-4320.

Förster B., Osmond C.B., Pogson B.J. (2005) Improved survival of very high light and oxidative stress is conferred by spontaneous gain-of-function mutations in Chlamydomonas. Biochimica et Biophysica Acta-Bioenergetics 1709: 45-57.

A novel nutrient uptake mechanism in the intracellular malaria parasite

Three groups in the School, together with collaborators at the University of Melbourne, have published a paper in the top-ranked international science journal Nature. The paper describes a novel ‘transporter mechanism’ used by the single-celled malaria parasite to take up the essential nutrient, phosphate, from its host red blood cell. The mechanism is ‘energised’ by sodium ions which, as members of the team have shown previously, flood into the red blood cell through novel channels induced by the parasite in the surface membrane of the red blood cell.

Saliba, K.J.¹, Martin, R.E.¹, Bröer, A., Henry, R.I., McCarthy, C.S., Downie, M.J., Allen, R.J.W., Mullin, K.A., McFadden, G.I., Bröer, S.² and Kirk, K.² (2006)  Sodium-dependent uptake of inorganic phosphate by the intracellular malaria parasite.  Nature  443, 582-585. [1,2: Equal contributions] [PubMed]

Splenic endothelial cells support development of dendritic cells from bone marrow.

The development of dendritic cells (DC) supported by splenic stroma has been studied in this laboratory since the early 90s. As a PhD student, Keping Ni was the first to develop a longterm culture system which continuously produced cells resembling DC. This finding occurred well before markers and antibodies were available to distinguish this cell type. Over time, the maintenance of progenitors within a stromal matrix became evident. A number of studies then showed the production of immature myeloid DC evident by cell surface marker expression, immune function, antigen presenting capacity, and later gene expression profiling. The importance of stromal cells in DC hematopoiesis was also revealed. Studies on stromal components were only possible after the STX3 stromal cell line was developed from one longterm culture which had by chance lost hematopoietic cells over time and cell passage. STX3 has since been used as a valuable tool in studies on the stromal cell components which influence DC hematopoiesis. For example, overlaid bone marrow or spleen cells produce DC with 14 days of coculture. These DC resemble those produced in longterm spleen cultures, with production of no other hematopoietic cell types. STX3 is, however, a heterogeneous stromal line, and so studies have been limited by the interaction effects of several different cell types within stroma.

This paper now describes the results of cloning STX3, and the range of different cell types isolated. Despite a common endothelial origin, the cloned lines were found to vary in hematopoietic support capacity for DC development. Some lines were identified as supporters and some as non-supporters. This study highlights the importance of vascular niches in hematopoiesis particularly in the context of the spleen microenvironment. It also emphasises the need to study the development and heterogeneity of cells of the endothelial lineage.

Figure shows the heterogeneity evident amongst cloned stromal cell lines.

Despars, G.D. and O'Neill, H.C.(2006) Splenic endothelial cells support development of dendritic cells from bone marrow. Stem Cells, 24, 1496-1504. [PubMed]

Bacteria manipulate plant hormones

Plants are in constant contact with soil bacteria, some of which are pathogenic, some symbiotic. Certain soil bacteria invade plant roots and form nodules in which they fix nitrogen, providing the plant with free biological fertilizer. So far it is not clear how the bacteria alter plant development to make a now root organ. We have been investigating whether these bacteria manipulate plant development by interfering with the plant's own growth hormones, in particular with auxin.

Our findings showed that nitrogen fixing bacteria altered the transport of auxin not just locally, where they invade the root, but even over long distances in the leaves of the plant. By modifying how much of the growth hormones flow from the leaves to the roots, the numbers of nodules on the root system can be modified. In search for a secondary signal that the bacteria use to modify auxin transport, we used RNA interference to silence the flavonoid pathway in a legume plant. Flavonoids have been suggested as potential auxin transport inhibitors. Our study provides the first genetic evidence that flavonoids in the plant are necessary for the regulation of auxin transport during the development of a nodule.

Prayitno J, Rolfe B.G., Mathesius U. (2006) The ethylene insensitive sickle mutant of Medicago truncatula shows altered auxin transport regulation during nodulation. Plant Physiology , in press (published online 14/07/2006 ).

Wasson, A.P., Pellerone, F.I. and Mathesius U. (2006) Silencing the flavonoid pathway in Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia . Plant Cell 18, 1617-1629. (This paper is accompanied by an editorial "In this issue" Plant Cell 18: 1539-1540.)

Van Noorden, G.E., Ross, J.J, Reid, J.B., Rolfe, B.G. and U. Mathesius (2006) Defective long distance auxin transport regulation in the Medicago truncatula super numeric nodulation mutant. Plant Physiology 140: 1494-1506

The acidity of the malaria parasite’s internal ‘digestive vacuole’

As the malaria parasite grows inside its host red blood cell it sucks up much of the host cell’s haemoglobin (the protein used by red blood cells to carry oxygen around the body). The haemoglobin is deposited in an internal acidic compartment, the parasite’s ‘digestive vacuole’, where it is chopped up into small pieces. A number of antimalarial drugs, including the widely used drug chloroquine, accumulate in the digestive vacuole, and this is where they are thought to exert their toxic effect.

The acidity of the digestive vacuole plays a key role in the accumulation of drugs, and it has also been suggested that changes in the acidity might contribute to the phenomenon of chloroquine resistance. Measuring the acidity (i.e. the pH) of the vacuole is technically very difficult. In this study we have loaded fluorescent dyes into the vacuole then used these dyes to estimate the pH in both a chloroquine-sensitive and a chloroquine-resistant parasite strain. We conclude that the pH in the parasite’s digestive vacuole is in the range 4.5-4.9, and that it is approximately the same in the chloroquine-sensitive and chloroquine resistant parasites. The primary explanation for chloroquine resistance must therefore lie elsewhere.

Hayward, R., Saliba, K.J. and Kirk, K. (2006) The pH of the digestive vacuole of Plasmodium falciparum is not associated with chloroquine resistance. J. Cell Science, 119, 1016-1025. [PubMed]

With the emergence and spread of malaria parasites that are resistant to most of the antimalarial drugs that we currently have available, there is an urgent need to develop new strategies to kill the parasite. It has been known for a long time that in order for malaria parasites to survive within the red blood cells of the human host they need to acquire vitamin B5 from the blood plasma. Interfering with the parasite’s ability to use vitamin B5, therefore, represents an attractive way by which malaria parasites might be eliminated.

We have identified two compounds, provitamin B5 and CJ-15,801, which are structurally similar to, but functionally different from, vitamin B5 (i.e. they cannot work as vitamin B5 in malaria parasites). These compounds have been shown to be effective at killing malaria parasites, and to do so by specifically interfering with the parasite’s ability to utilise vitamin B5. We also found that these compounds are relatively non-toxic to mammalian cells, opening the door to using this class of compounds as antimalarials.

A commentary on aspects of this work has been published in Drug Discovery Today, 2005, 10: 447-448.

Saliba, K.J., Ferru, I. and Kirk, K. (2005) Provitamin B5 (pantothenol) inhibits growth of the intraerythrocytic malaria parasite. Antimicrob. Agents Chemother., 49, 632-637

Saliba, K.J. and Kirk, K. (2005) CJ-15,801, a fungal natural product, inhibits the intraerythrocytic stage of Plasmodium falciparum in vitro via an effect on pantothenic acid metabolism. Molec. Biochem. Parasitol., 141: 129-131.

Iminoglycinuria is an inherited disorder affecting the absorption of amino acids in the kidneys and the small intestine. Its diagnostic hallmark is a striking increase of proline and glycine in the urine. The phenotype of the disorder is variable, including observations of isolated loss of glycine and cases where intestinal amino acid transport is not affected.

The disease was first described in 1968 and it was soon proposed that mutations in an amino acid transporter may underlie the disease. In our studies we have identified a novel transporter (SLC6A20) isolated from mouse and human kidney, which shows properties of the transporter affected in Iminoglycinuria.

Kowalczuk, S., Broer, A., Munzinger, M., Tietze, N., Klingel, K. and Broer, S. (2005) Molecular cloning of the mouse IMINO system, a Na⁺ and Cl^- - dependent proline transporter. Biochem, J., online.

The image shows the presence of mRNA for this transporter in kidney cortex.

Research over recent years has identified two proteins which, when mutated, can influence the degree of resistance of the malaria parasite to the antimalarial drug chloroquine. One of these proteins, the ‘chloroquine resistance transporter’ is discussed below (see “Learning about the malaria parasite’s ‘chloroquine resistance transporter’ from others in its family”). The other is called ‘P-glycoprotein homologue 1’, or Pgh1 for short. How mutations in this protein influence the sensitivity of the parasite to antimalarial drugs is still unclear. But what we have shown in this study is that mutations in Pgh1 that increase the degree of chloroquine resistance, also have the effect of reducing the overall ‘vigour’ of the parasite, retarding its growth relative to parasites that retain the ‘normal’ form of the protein.

High levels of chloroquine resistance therefore come at a significant cost to the malaria parasite.

Hayward, R., Saliba, K.J. and Kirk, K. (2005) pfmdr1 mutations associated with chloroquine resistance incur a fitness cost in Plasmodium falciparum. Molecular Microbiology, 55, 1285-1295.

Chloroquine, an antimalarial developed during the 1930s, was in many senses a 'wonder drug' - cheap, effective, and with minimal side effects. By the 1950s, however, malaria parasites emerged that were resistant to chloroquine. Since then chloroquine-resistant parasites have spread slowly but surely throughout endemic areas, and the drug is now virtually useless in the places where it is most-needed.

Chloroquine resistance arises as a result of mutations in a protein christened the 'chloroquine resistance transporter'. This protein sits in the membrane enclosing an internal compartment in the parasite, though what it is doing there, and how changes in this protein confer chloroquine resistance to the parasite is unclear. In this 'bioinformatic' study we have shown that the protein is actually a member of a large family of proteins which, in other organisms have the job of moving small molecules across membranes. Other members of the family have the same general shape and configuration as the chloroquine resistance transporter, and it is clear from studies on other family members that the changes that give rise to chloroquine resistance are in a part of the protein that determines the size, shape and electrical charge of the molecules that that protein can carry across the membrane. This has led to the hypothesis that mutations in the chloroquine resistance transporter change the protein in such a way as to allow it to carry chloroquine out of the compartment in which it exerts its antimalarial effect, thereby decreasing the effectiveness of the drug.

This paper has attracted a number of commentaries (Drug Discovery Today, 9, 814-5; Faculty of 1000).

Martin, R.E. and Kirk, K. (2004) The malaria parasite's chloroquine resistance transporter is a member of the drug/metabolite transporter superfamily. Molecular Biology and Evolution, 21: 1938-1949.

Using a novel patented technology for anchoring targeting molecules onto lipid membranes, researchers at the ANU have shown that they can target antigen-containing stealth liposomes and tumour-derived plasma membrane vesicles directly to dendritic cells, to enhance the body’s immune response against cancer.

This novel type of vaccine which targets the body’s dendritic cells was tested in mice. The vaccine induced strong anti-tumuor responses and therapeutic effect in mice bearing the highly metastatic B16-OVA melanoma, prolonging their disease-free survival. The targeting of antigen to DCs in this way could be an effective procedure for enhancing tumour immunity and immunotherapy, and plans are underway to test this approach in patients with metastatic melanoma. The work is being sponsored by Lipotek Pty Ltd.

van Broekhoven, C.L., Parish, C.R., Demangel, C., Britton, W.J. and Altin, J.G. (2004) Targeting dendritic cells with antigen-containing liposomes: a highly effective procedure for induction of anti-tumor immunity and for tumor immunotherapy. Cancer Research 64, 4357-4365. (Cover feature and research paper).

Hartnup disorder is an inherited disorder affecting the absorption of amino acids in the kidneys and the small intestine. Its diagnostic hallmark is a striking increase of neutral amino acids in the urine. Other symptoms reported in Hartnup disorder patients include a photosensitive skin rash, difficulties to control gait and psychotic behaviour.

The disease was first described in 1956 and it was soon proposed that mutations in an amino acid transporter may underlie the disease. In our studies we have identified a novel transporter (SLC6A19) isolated from mouse and human kidney, which shows all properties of the transporter affected in Hartnup disorder. Sequencing of the SLC6A19 gene from Hartnup patients subsequently demonstrated that the disorder is indeed caused by mutations in this gene.

The image shows the presence of mRNA for this transporter in the brush border of the small intestine. The mRNA is detected by a radioactive probe, which binds to the RNA. Black grains indicate the presence of mRNA for the transporter.

ABC News Story

Seow, H. F., Broer, S., Broer, A., Bailey, C. G., Potter, S. J., Cavanaugh, J. A. and Rasko, J. E. (2004). Hartnup disorder is caused by mutations in the gene encoding the neutral amino acid transporter SLC6A19. Nature Genetics, 36 (9): 1003-7. Online

Bröer, A., Klingel, K., Kowalczuk, S., Rasko, J.E., Cavanaugh, J. and Broer, S. (2004) Molecular cloning of mouse amino acid transport system B0, a neutral amino acid transporter related to Hartnup disorder. J Biol Chem. 279 (23): 24467-24476.

There are approximately 165 million cases of shigellosis world wide annually, resulting in 1.1 million deaths. The majority of cases occur in developing countries and most deaths occur in children under 5 years of age.

Shigellosis is mainly caused by the bacterium Shigella flexneri. There are 13 different serotypes of S. flexneri determined by bacterial cell-surface sugar chains called O-antigens. Bacterial viruses (bacteriophages) carry the genes which confer O-antigen variation. Infection and subsequent incorporation of the virus into the genetic material of the bacterial cell results in modification of the bacterial O-antigen. We are interested in studying fundamental processes related to the O-antigen modification. Our group has shown that three enzymes are involved in this process and all of them are found to be integral membrane proteins. We have recently determined the topological structure of one of these proteins encoded by the O-antigen modification gene cluster. This study will allow us to further investigate how these proteins interact with each other and function in the membrane of Shigella bacteria.

Korres, H. and Verma, N. K. (2004) Topological analysis of glucosyltransferase GtrV of Shigella flexneri by a dual reporter system and identification of a unique reentrant loop. Journal of Biological Chemistry 279, 22469-22476.

Following on from our analysis of the size and origin of the parasite’s ‘membrane potential’ we have shown that it plays a key role in the uptake by the parasite of at least one important nutrient. Choline is a small, positively charged molecule, used by the parasite in the synthesis of new membranes. We have shown that choline is taken up by the parasite, across its surface membrane, via a ‘transporter’ that is energised by the transmembrane voltage. The electrical properties of the parasite membrane are therefore of fundamental importance in the biology of this important organism, and the mechanisms underlying these properties may be suitable antimalarial drug targets.

Allen, R.J.W. and Kirk, K. (2004) The membrane potential of the intraerythrocytic malaria parasite, Plasmodium falciparum. J. Biol. Chem., 279, 11264-11272.