Current position
Professor and Group Leader, Plant Cell Biology Group, RSBS, ANU.
Plant-pathogen interactions: the infection of plants by Oomycetes and fungi
Professor Adrienne R. Hardham
Overview of research program:
 Research aims: The aim of our research is to elucidate the cellular and molecular basis of the infection of plants by Oomycetes and fungi, with a particular focus on Phytophthora, a genus of highly destructive plant pathogens. We employ a fully integrated approach that uses the latest techniques in molecular genetics and genomics, proteomics, cell biology and microscopy.
Economic and environmental impact of Phytophthora diseases: Phytophthora and other Oomycetes cause widespread and devastating diseases in important agricultural crops and natural ecosystems. P. infestans, for example, causes Late Blight of potato; P. sojae causes root rot of soybean; P . nicotianae is a major pathogen of citrus; and P. cinnamomi kills thousands of plants, including an alarming number of species in native forests and heathlands in southern Australia. Many aspects of the development and pathogenicity of Phytophthora and other Oomycetes are similar to those of fungi, but they are phylogenetically quite distinct from true fungi. Biochemical differences between Oomycetes and true fungi mean that Oomycetes are not inhibited by many of the chemicals used to control fungal diseases.
Phytophthora infection cycle: Phytophthora species, like other Oomycetes, form sexual and asexual spores. For most species the major infective agents are motile, biflagellate zoospores that are chemotactically attracted to potential infection sites on roots and leaves. Zoospores and hyphae secrete adhesive material that attaches the pathogen cells to the plant host. Hyphae secrete a wide range of virulence factor s including enzymes that degrade the plant cell wall.
Phytophthora life cycle diagram, light micrograph of zoospore release from a sporangium lower right), and scanning electron micrograph of a biflagellate zoospore (left).
Current projects in our research program:
Our research aims to identify and characterise Phytophthora proteins that play key roles in the infection of plants. We use shot-gun monoclonal antibody production, proteomics and cDNA/EST libraries to identify stage-specific proteins; we use immunocytochemistry, GFP-tagging and gene silencing to determine their localisation and function.
 1. Zoospore motility (Dr Leila Blackman, Professor Toshinobu Suzaki and Dr John Harper): The ability of biflagellate zoospores to swim chemotactically to suitable infection sites is a key feature of the Phytophthora infection strategy. We have shown that tubular hairs called mastigonemes on the anterior flagellum contain a 40 kDa glycoprotein (Robold and Hardham 1998) and that they are responsible for forward motion of the spores (Cahill et al. 1996). In a collaborative project with Professor Toshinobu Suzaki and Shuhei Yamada at Kobe University in Japan, we have cloned the gene encoding the mastigoneme protein and are currently characterising the small gene family to which it belongs. Our analysis of zoospore motility also includes studies of the calcium-sensitive, centrin, which forms a number of contractile fibres in the flagellar apparatus (Harper et al. 1995) and serves a variety of roles during the cell cycle. We are currently studying the composition, expression and function of members of the Phytophthora centrin gene family.
 
2. Zoospore adhesion to the host plant surface (Dr Leila Blackman, Ms Victoria Ludowici): Spore attachment to the host surface is an important factor in the infection of plants. We were the first to clone a gene (PcVsv1) encoding a fungal/Oomycete spore adhesive protein (Robold and Hardham 2005) and discovered that it contained 47 copies of a TSR1 motif found in adhesives in animal cells and malarial parasites. We are currently investigating other proteins that are synthesised during sporulation and that may be packaged into the spore adhesive vesicles. We are also using gene silencing to determine the role of adhesion in the establishment of infection.
3. Complement control proteins and zoospore-specific gene expression (Dr Leila Blackman, Ms Victoria Ludowici): Differential screening of a cDNA library constructed from mRNA isolated from Phytophthora zoospores has revealed the presence of many gene transcripts that are specifically or preferentially expressed in motile zoospores (Škalamera et al. 2004). One of these genes encodes a protein containing the complement control protein module (Škalamera and Hardham 2006). The protein, PnCcp, contains a signal sequence directing insertion into the secretory pathway and PnCcp is packaged into vesicles in the asexual spores. We have generated transformants in which expression of PnCcp is silenced and are currently studying the effects of PnCcp down-regulation. Release of PnCcp during zoospore encystment has given evidence of selective secretion from a spore cortical vesicle and this unusual process is being investigated in detail.
  4. Use of transformation to study P. nicotianae genes preferentially expressed in germinated cysts (Dr Reena Narayan, Ms Soma Chakraborty, Dr Weixing Shan, Dr Leila Blackman): Protocols for the transformation of P. nicotianae have been developed (Bottin et al. 1999) and we are using them to introduce RNAi silencing and GFP-tagging constructs into P. nicotianae for functional genomic studies of genes preferentially expressed in germinated cysts (Shan et al. 2004), the developmental stage responsible for initial penetration of the plant. We have generated extensive resources with which to document gene transcript levels using real-time, quantitative RT-PCR and assays to test effects on pathogen development and virulence.
 5. Studies of the Phytophthora cyclophilin gene family (Ms Pamela Gan, Dr Weixing Shan, Dr Leila Blackman): Two genes that are highly expressed in germinated cysts encode members of the cyclophilin family of peptidyl-prolyl isomerases. These enzymes assist protein folding and contribute to a variety of processes in eukaryotic cells, including the virulence of fungal pathogens. We have used bioinformatics analyses to integrate the results of our genomic and expression studies of the P. nicotianae cyclophilin gene family with publicly-available genomic and EST data from P. infestans, P. sojae and P. ramorum to elucidate the composition of the gene family and patterns of expression of its members.
6. Characte risation of cell wall degrading enzymes (Dr Leila Blackman, Ms Trish Boyce): Secretion of cell wall degrading enzymes is a major factor in the penetration and colonisation of host plants by bacterial, fungal and Oomycete pathogens. We have shown that P. cinnamomi contains a very large polygalacturonase gene family, encoding enzymes that break down pectin in the plant cell wall (Götesson et al. 2002). The family contains over 20 members, and analysis of their sequences has given strong evidence of the operation of diversifying selection during pathogen evolution. A similar number of xyloglucan-specific endoglucanases has also been reported in P. infestans (Costanzo et al. 2006). The rationale for such large families of cell wall degrading enzymes is not fully understood. However, because the polysaccharides forming plant cell walls are highly complex and diverse, it may be that large gene families allow enzyme specialisation to target polysaccharides with particular structures. Patterns of expression of pathogen cell wall degrading enzymes are complex and there is evidence of expression cascades and catabolite  repression. We are currently investigating patterns of expression of polygalacturonase genes during in vitro and in planta growth of P. cinnamomi and P. nicotianae, with a view to fully elucidating their role in pathogen virulence.
  7. Cell biology of the plant response to Oomycete pathogens (Dr Daigo Takemoto, Dr Rosemary White, Dr David Jones): Plants can respond very quickly to attempted infection by Oomycete and fungal pathogens (Hardham 2007). Rapid structural changes include cytoplasmic aggregation at the infection site and reorganisation of the plant cytoskeleton. Studies of the interaction of compatible, incompatible and non-adapted Oomycete pathogens with Arabidopsis plants in which components of the cytoskeleton and endomembrane system had been tagged with GFP, has revealed that early structural changes are similar in all three categories of interaction (Takemoto et al. 2003, Takemoto et al. 2006, Takemoto and Hardham 2004). In all cases, actin microfilaments become focused underneath the invading pathogen and are responsible for moving cytoplasmic components to the infection site. The ubiquity of this response suggests a non-specific trigger and we are currently investigating the effects of physical and chemical stimuli on the reorganisation of cytoskeletal and endomembrane elements in the plant cells.
8. Elucidation of pathogen effector uptake by plants (Dr Mayam Rafiqu, Ms Pamela Gan, Mr Markus Koeck, Dr Peter Dodds, Dr Jeff Ellis, Dr David Jones): One of the most exciting recent developments in our understanding of plant-pathogen interactions has been the realization that fungal and Oomycete pathogens deliver small effector proteins into the cytoplasm of host cells (Catanzariti et al. 2006, Dodds et al. 2004, Kemen et al. 2005). This insight stems from the identification of pathogen avirulence proteins that are recognized by host resistance proteins inside plant cells as part of the host defence response. Indeed, intracellular delivery of effector proteins is likely to be the key process facilitating pathogen manipulation of the host during infection. In a collaborative project with colleagues in the Plant Cell Biology Group (Dr David Jones) at CSIRO (Drs Peter Dodds and Jeff Ellis), we are investigating the molecular basis of fungal effector protein transport into host plant cells and their function as virulence determinants or as avirulence factors that induce host defences.
References Cited
Bottin A, Larche L, Villalba F, Gaulin E, Esquerré-Tugayé M-T, Rickauer M (1999) Green fluorescent protein (GFP) as gene expression reporter and vital marker for studying development and microbe-plant interaction in the tobacco pathogen Phytophthora parasitica var. nicotianae. FEMS Microbiol Lett 176: 51-56
Cahill DM, Cope M, Hardham AR (1996) Thrust reversal by tubular mastigonemes: immunological evidence for a role of mastigonemes in forward motion of zoospores of Phytophthora cinnamomi. Protoplasma 194: 18-28
Catanzariti A-M, Dodds PN, Lawrence GJ, Ayliffe MA, Ellis JG (2006) Haustorially expressed secreted proteins from flax rust are highly enriched for avirulence elicitors. Plant Cell 18: 1-14
Costanzo S, Ospina-Giraldo MD, Deahl KL, Baker CJ, Jones RW (2006) Gene duplication event in family 12 glucosyl hydrolase from Phytophthora spp. Fung Genet Biol 43: 707-714
Dodds PN, Lawrence GJ, Catanzariti A-M, Ayliffe MA, Ellis JG (2004) The Melampsora lini AvrL567 avirulence genes are expressed in haustoria and their products are recognized inside plant cells. Plant Cell 16: 755-768
Götesson A, Marshall JS, Jones DA, Hardham AR (2002) Characterization and evolutionary analysis of a large polygalacturonase gene family in the oomycete plant pathogen Phytophthora cinnamomi. Mol Plant-Microbe Interact 15: 907-921
Hardham AR (2007) Cell biology of plant-oomycete interactions. Cellular Microbiology 9: 31-39
Harper JDI, Gubler F, Salisbury JL, Hardham AR (1995) Centrin association with the flagellar apparatus in spores of Phytophthora cinnamomi. Protoplasma 188: 225-235
Kemen E, Kemen AC, Rafiqi M, Hempel U, Mendgen K, Hahn M, Voegele RT (2005) Identification of a protein from rust fungi transferred from haustoria into infected plant cells.
Mol Plant-Microbe Interact 18: 1130-1139
Robold AV, Hardham AR (1998) Production of species-specific monoclonal antibodies that react with surface components on zoospores and cysts of Phytophthora nicotianae. Can J Microbiol 44: 1161-1170
Robold AV, Hardham AR (2005) During attachment Phytophthora spores secrete proteins containing thrombospondin type 1 repeats. Curr Genet 47: 307-315
Shan W, Marshall JS, Hardham AR (2004) Gene xpression in germinated cysts of Phytophthora nicotianae. Molec Plant Pathol 5: 317-330
Škalamera D, Hardham AR (2006) PnCcp, a Phytophthora nicotianae protein containing a single complement control protein module, is sorted into large peripheral vesicles in zoospores. Aust Plant Pathol 35: 593-603
Škalamera D, Wasson AP, Hardham AR (2004) Genes expressed in zoospores of Phytophthora nicotianae. Molecular Genetics & Genomics: MGG 270: 549-557
Takemoto D, Hardham AR (2004) Update: The cytoskeleton as a regulator and target of biotic interactions in plants. Plant Physiol 136: 3864-3876
Takemoto D, Jones DA, Hardham AR (2003) GFP-tagging of cell components reveals the dynamics of subcellular re-organization in response to infection of Arabidopsis by oomycete pathogens. Plant J 33: 775-792
Takemoto D, Jones DA, Hardham AR (2006) Re-organization of the cytoskeleton and endoplasmic reticulum in the Arabidopsis pen1-1 mutant inoculated with the non-adapted powdery mildew pathogen, Blumeria graminis f. sp hordei. Molec Plant Pathol 7: 553-563
Other publications during the last 5 years:
Ambikapathy J, Marshall JS, Hocart CH, Hardham AR (2002) The role of proline in osmoregulation in Phytophthora nicotianae. Fung Genet Biol 35: 287-299
B
lackman, L.M., Mitchell, H.J. and Hardham, A.R. (2005) Characterisation of manganese superoxide dismutase from Phytophthora nicotianae. Mycological Research 109, 1171-1183.
Blackman L.M. and Hardham, A.R. (2008) Regulation of catalase activity and gene expression during Phytophthora nicotianae development and infection of tobacco. Molecular Plant Pathology. In press.
Frost, D., Way, H., Howles, P., Luck, J., Manners, J., Hardham, A. and Finnegan, J. (2004) Tobacco transgenic for the flax rust resistance gene L expresses allele-specific activation of defence responses. Molecular Plant-Microbe Interactions 17, 224-232.
Hardham, A.R. (2004) Fungal and Oomycete Plant Pathogens: Cell Biology. Encycl. Plant & Crop Science 480-483.
Hardham, A.R. (2004) Functioning Cells. In: Biology 3rd Edition. Eds P. Ladiges, B. Evans and R. Saint. pp.69-105. McGraw-Hill Book Company, Sydney.
Hardham AR (2005) Pathogen profile: Phytophthora cinnamomi. Molec Plant Pathol 6: 589-604
Hardham, A.R. (2007) Cell biology of fungal and oomycete infection of plants. In The Mycota Vol VIII: Biology of the Fungal Cell, Second Edition, edited by R.J. Howard and N.A.R. Gow. Springer-Verlag. Pp. 251-289.
Hardham, A.R. and Shan, W. (2008) Cellular and molecular biology of Phytophthora-plant interactions. The Mycota Vol. V. Plant Relationships, Second Edition, edited by H. Deising. Springer-Verlag. In press.
Hardham, A.R. and Takemoto, D. (2006) Dynamic subcellular responses in plants during interactions with fungi and Oomycetes. In Biology of plant-microbe interactions Volume 5. pp. 70-79. ISMP-MI, St Paul, MN.
Hardham, A.R., Takemoto, D. and Jones, D.A. (2007) Cytoskeleton and cell wall function in penetration resistance. Current Opinion in Plant Biology 10, 342-348.
Jiang RHY, Tyler BM, Whisson SC, Hardham AR, Govers F (2005) Ancient origin of elicitin gene clusters in Phytophthora genomes. Mol Biol Evol 23: 338-351
Mitchell HJ, Kovac KA, Hardham AR (2002) Characterisation of Phytophthora nicotianae zoospore and cyst membrane proteins. Mycol Res 106: 1211-1223
Shan W, Hardham AR (2004) Construction of a bacterial artificial chromosome library, determination of genome size, and characterization of an Hsp70 gene family in Phytophthora nicotianae. Fung Genet Biol 41: 369-380
Shan W, Liu J, Hardham AR (2006) Phytophthora nicotianae PnPMA1 encodes an atypical plasma membrane H+-ATPase that is functional in yeast and developmentally regulated. Fung Genet Biol 43: 583-592
Takemoto D, Hardham AR, Jones DA (2005) Differences in cell death induction by phytophthora elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus. Plant Physiol 138: 1491-1504
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