Presidential Address: Plant-pathogen interactions: advancing understanding
of plants and microbes
Research into plant-pathogen interactions generates greater understanding of essential metabolic activities in plants and microbes. Examples will be given of how such added value has stemmed from work on a range of plant diseases. Topics to be covered include, 1) phytoalexins, their biosynthetic pathways and detoxification; 2) localized defences to colonisation and their cellular co-ordination; 3) Hrp genes, Type III secretion and protein injection by bacteria; 4) the HR, calcium and co-ordination of the oxidative burst, and 5) avr, vir and R genes and molecular recognition. The new insights gained from work in these areas will be highlighted and opportunities for the development of new targets for disease control discussed.
Session I: The Structural Framework
Life at the edge: the tobacco mosaic virus-induced hypersensitive response
The incompatible interaction between tobacco mosaic virus (TMV) and Nicotiana plants carrying the N gene results in a hypersensitive response (HR) with the development of necrotic local lesions at the sites of virus invasion and containment of the pathogen. In order to study the cellular events preceding the appearance of visible symptoms we have used a tagged TMV genome expressing the green fluorescent protein. Using a temperature shift, from 33 C to 20 C, to initiate and synchronise the HR we have monitored presymptomatic infection foci by fluorescence, confocal and electron microscopy. Under these conditions cell death occurs approximately 12 hours after temperature shift with the initial 6 hours representing the activation phase and the subsequent 6 hours representing the execution phase of the HR. Following cell death and necrotic lesion development virus infected cells are still present at the necrotic lesion margin, however, all remaining infected cells eventually die indicating that for this pathosystem the HR is a two phase process.
In parallel studies aimed at investigating the induction of cell death in plants by the mammalian proapoptotic protein bax a number of similarities to the TMV-induced hypersensitive response were observed suggesting the possibility that bax could interact with components of the plant cell death machinery. Significantly the cytotoxic effect of bax required mitochondrial localisation raising the possibility that in plants, like animals, cell death signalling is transduced through mitochondria. To investigate this possible link between plant and animal cell death systems both electron microscopy and immunochemistry were used to study mitochondria during the early, presymptomatic, activation and execution phases of the HR.
The infection process of the bean anthracnose fungus Colletotrichum
Colletotrichum species cause anthracnose on a wide range of plants. During infection of leaves, the pre-penetration events are generally similar between species, however post-penetration development can be either subcuticular or hemibiotrophic. Colletotrichum lindemuthianum is in the latter grouping, and aspects of spore adhesion, germination and appressorium development will be described as well as the formation of biotrophic and necrotrophic hyphae.
Function of pili in molecular interaction of Pseudomonas syringae
with the host plant
Pseudomonas syringae is an opportunistic plant pathogen which is capable of colonizing aerial plant surfaces and leaf tissue without causing disease symptoms. The bacterium possesses a variety of traits that makes it withstand the variable and harsh conditions on plant leaf surfaces. It is resistant to UV radiation, drought, and to dislocation by physical forces such as rain and wind. It is capable of taking advantage of rapid changes is the environmental conditions; upon rain it enters a phase of rapid growth, and in the presence of a susceptible host plant and conditions favouring disease outbreak it enters a pathogenic growth and gene-expression phase. In dry conditions on the leaf surface clusters of bacteria are found encapsulated in extracellular polysaccharide. At both growth phases epiphytic and pathogenic bacterial proteinaceous appendages called pili or fimbriae play a role in the plant-microbe interaction.
Genes that are important for the fitness of the bacterium during different growth phases thus include two gene-clusters that form extracellular appendages called pili. The type IV pilus is produced by genes that are related to the type II protein secretion pahtway. The type IV pilus is present in also many other plant related bacteria, and it promotes attachment of bacterial cells to leaf surfaces, auto-aggregation of bacteria, and possibly twitching motility. Attachment to surfaces and auto-aggregation both contribute to UV resistance and resistance against being washed away. Production of EPS apparently works in the same direction. When free water (rain, dew) becomes available on plant surfaces the bacteria rapidly take advantage e.g. by spreading, in part facilitated by flagellar motility. In this process it appears that they enter and exit substomatal cavities. Colonization data suggest that a large portion of the bacterial leaf surface population has its source inside the leaf tissue even in situation where no disease is observed.
The second pilus-producing gene-cluster is the hrp (hypersensitivity reaction and pathogenicity) gene cluster that is absolutely required for disease. Part of the gene cluster constitutes a type III protein secretion pathway, used for delivery of virulence/avirulence (Avr) determinants directly into the target cell cytoplasm. The Hrp-pilus mainly formed by units of HrpA is essential for disease apprently by being required in forming a link between bacterial (donor) and host plant (recipient) cells during translocation of virulence determinants. The Hrp-pilus may, however, also have functions related to bacterial attachment and aggregation. Contructs with the gene for green fluorescent protein (GFP) driven in a hrp- dependent manner were made. These constructs are used to study induction timing and localization, one of the questions being: is the Hrp-pilus present on the plant leaf surface? And if so, what might the role of the Hrp pilus, and the hrp gene cluster in general be in non-symptom generating P. syringae colonization? Preliminary data suggest that strong induction of hrp genes take place only inside the plant leaf tissue, but may clearly precede, and may not directly induce symptom development.
The current working model used to our further studies postulates that the Hrp-pilus has a direct and active function in translocating effector proteins into the plant cells. Whether the Hrp-pilus grows from the tip or the base, and whether the Hrp pilus is hollow, forming a conduit trough which (unfolded) proteins may be transported are among questions currently under investigation.
Conidial germination and germling development by Blumeria graminis:
from first touch to attempted penetration
Blumeria graminis (syn. Erysiphe graminis) causes cereal powdery mildew. On suitable substrata (e.g. host leaf), conidia germinate and germling development follows an orderly sequence. A short primary germ tube (PGT; emerging ca 30-90 min) performs at least three functions: adhesion, accessing water and recognising substratum characteristics. The latter drives elongation of a second-formed germ tube (to ca 40 μm) as a pre-requisite to appressorial differentiation. However, the elongate tube remains undifferentiated unless it in turn recognises substratum characteristics and responds by forming an apical appressorial lobe (by 8-12 h) from which a peg attempts penetration . To make substratum contact, germ tubes must emerge from the conidial wall close to the substratum interface. Around 80% or more of first-formed germ tubes make contact, and this is up to 8 times more frequently than predicted by chance. Experimentation showed the site of germ tube emergence to be determined within 1-3 min after deposition, stimulated by a very small contact interface and to be a relatively non-specific response to contact with host leaf and artificial substrata . Recent data implicate extracellular materials (ECM), released from conidial wall projections contacting the substratum, in directing the site of germ tube emergence. ECM, which can be released within 20 sec , may also be involved in relatively weak but rapid adhesion, prior to germ tube formation. Use of artificial and natural substrata indicates that PGTs and elongating appressorial germ tubes recognise a range of factors including substratum hydrophobicity, cutin monomers (released by fungal cutinase activity?) and possibly cellulose breakdown products (released by fungal cellulase activity?) . This knowledge has facilitated studies of the cell biological control of germling development (see Gurr, this meeting) and identifying potential targets for intervention through the development of novel fungicides or plant breeding. Indeed, natural phenomena can impede germling development: second-formed germ tubes frequently fail to elongate on abaxial surfaces of Lolium leaves, and factors released within established oat mildew colonies (but not barley mildew) cause failure of appressorial differentiation by elongating second-formed germ tubes.
1. Carver TLW, Ingerson SM, Thomas BJ. (1996). Influences of host surface features on development of Erysiphe graminis and Erysiphe pisi. In: Kerstiens, G. ed. Plant Cuticles- an integrated functional approach. Bios Scientific Publishers. Oxford. pp 255-266.
2. Wright AJ, Carver TLW, Thomas BJ, Fenwick NID, Kunoh H, Nicholson RL. (in press). The rapid and accurate determination of germ tube emergence site by Blumeria graminis conidia. Physiological and Molecular Plant Pathology.
3. Carver TLW, Kunoh H, Thomas BJ, Nicholson RL (1999). Release and visualization of the extracellular matrix of conidia of Blumeria graminis. Mycological Research 103: 547-560.